Plasmid maintenance system for antigen delivery

ABSTRACT

The present invention relates generally to a Plasmid Maintenance System for the stabilization of expression plasmids encoding foreign antigens, and methods for making and using the Plasmid Maintenance System. The invention optimizes the maintenance of expression plasmids at two independent levels by: (1) removing sole dependence on balanced lethal maintenance functions; and (2) incorporating at least one plasmid partition function to prevent random segregation of expression plasmids, thereby enhancing their inheritance and stability. The Plasmid Maintenance System may be employed within a plasmid which has been recombinantly engineered to express a variety of expression products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation Application of application Ser. No.10/750,976, filed Jan. 5, 2004, now U.S. Pat. No. 6,977,176, which is aDivisional of application Ser. No. 09/453,313 (now U.S. Pat. No.6,703,233), filed Dec. 2, 1999, which is a Continuation-in-PartApplication of U.S. application Ser. No. 09/204,117, filed Dec. 2, 1998(now U.S. Pat. No. 6,413,768), and also claims benefit of U.S.Provisional Application No. 60/158,738, filed Oct. 12, 1999. Each ofthese prior applications is hereby incorporated by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with support pursuant to National Institutes ofHealth Grant No. RO1 AI29471. The government may have certain rights inthis invention.

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to expression plasmidsstabilized by a Plasmid Maintenance System (as defined herein) capableof expressing a protein or peptide, such as an antigen for use in a livevector vaccine, and methods for making and using the stabilizedplasmids. The invention optimizes the maintenance of expression plasmidsat two independent levels by: (1) removing sole dependence on catalyticbalanced lethal maintenance systems; and (2) incorporating a plasmidpartition system to prevent random segregation of expression plasmids,thereby enhancing inheritance and stability.

1.2 Description of Related Art

Set forth below is a discussion of art relevant to the presentinvention.

1.2.1 Bacterial Live Vector Vaccines

Bacterial live vector vaccines deliver antigens to a host immune systemby expressing the antigens from genetic material contained within abacterial live vector. The genetic material is typically a replicon,such as a plasmid. The antigens may include a wide variety of proteinsand/or peptides of bacterial, viral, parasitic or other origin.

Among the bacterial live vectors currently under investigation areattenuated enteric pathogens (e.g., Salmonella typhi, Shigella, Vibriocholerae), commensals (e.g., Lactobacillus, Streptococcus gordonii) andlicensed vaccine strains (e.g., BCG). S. typhi is a particularlyattractive strain for human vaccination.

1.2.2 Attenuated Salmonella typhi as a Live Vector Strain

S. typhi is a well-tolerated live vector that can deliver multipleunrelated immunogenic antigens to the human immune system. S. typhi livevectors have been shown to elicit antibodies and a cellular immuneresponse to an expressed antigen. Examples of antigens successfullydelivered by S. typhi include the non-toxigenic yet highly immunogenicfragment C of tetanus toxin and the malaria circumsporozoite proteinfrom Plasmodium falciparum.

S. typhi is characterized by enteric routes of infection, a qualitywhich permits oral vaccine delivery. S. typhi also infects monocytes andmacrophages and can therefore target antigens to professional APCs.

Expression of an antigen by S. typhi generally requires incorporation ofa recombinant plasmid encoding the antigen. Consequently, plasmidstability is a key factor in the development of high quality attenuatedS. typhi vaccines with the ability to consistently express foreignantigens.

Attenuated S. typhi vaccine candidates for use in humans should possessat least two well separated and well defined mutations thatindependently cause attenuation, since the chance of in vivo reversionof such double mutants would be negligible. The attenuated vaccinecandidate S. typhi CVD908 possesses such properties. CVD908 contains twonon-reverting deletion mutations within the aroC and aroD genes. Thesetwo genes encode enzymes critical in the biosynthetic pathway leading tosynthesis of chorismate, the key precursor required for synthesis of thearomatic amino acids phenylalanine, tyrosine, and tryptophan. Chorismateis also required for the synthesis of p-aminobenzoic acid; after itsconversion to tetrahydrofolate, p-aminobenzoic acid is converted to thepurine nucleotides ATP and GTP.

1.2.3 Plasmid Instability

Plasmidless bacterial cells tend to accumulate more rapidly thanplasmid-bearing cells. One reason for this increased rate ofaccumulation is that the transcription and translation of plasmid genesimposes a metabolic burden which slows cell growth and gives plasmidlesscells a competitive advantage. Furthermore, foreign plasmid geneproducts are sometimes toxic to the host cell.

Stable inheritance of plasmids is desirable in the field of attenuatedbacterial live vector vaccines to ensure successful continued antigenproduction, as well as in commercial bioreactor operations in order toprevent bioreactor takeover by plasmidless cells.

Stable inheritance of a plasmid generally requires that: (1) the plasmidmust replicate once each generation, (2) copy number deviations must berapidly corrected before cell division, and (3) upon cell division, theproducts of plasmid replication must be distributed to both daughtercells.

Although chromosomal integration of foreign genes increases thestability of such sequences, the genetic manipulations involved can bedifficult, and the drop in copy number of the heterologous gene oftenresults in production of insufficient levels of heterologous antigen toensure an optimal immune response. Introduction of heterologous genesonto multicopy plasmids maintained within a live vector strain is anatural solution to the copy number problem; genetic manipulation ofsuch plasmids for controlled expression of such heterologous genes isstraightforward. However, resulting plasmids can become unstable invivo, resulting in loss of these foreign genes.

1.2.4 Plasmid Stabilization Systems

In nature bacterial plasmids are often stably maintained, even thoughusually present at very low copy numbers. Stable inheritance ofnaturally occurring lower copy number plasmids can depend on thepresence of certain genetic systems which actively prevent theappearance of plasmid-free progeny. A recent review of plasmidmaintenance systems can be found in Jensen et al. Molecular Microbiol.17:205–210, 1995 (incorporated herein by reference).

1.2.5 Antibiotic Resistance

One means for maintaining plasmids is to provide an antibioticresistance gene on the plasmid and to grow the cells inantibiotic-enriched media. However, this method is subject to a numberof difficulties. The antibiotic resistance approach is expensive,requiring the use of costly antibiotics and, perhaps more importantly,the use of antibiotics in conjunction with in vivo administration ofvaccine vectors is currently discouraged by the U.S. Food and DrugAdministration.

In large-scale production applications, the use of antibiotics mayimpose other limitations. With respect to commercial bioreactors,antibiotic resistance mechanisms can degrade the antibiotic and permit asubstantial population of plasmidless cells to persist in the culture.Such plasmidless cells are unproductive and decrease the output of thebioreactor.

There is therefore a need in the art for a plasmid maintenance systemspecifically designed for use in bacterial live vector vaccines whichdoes not rely on antibiotic resistance, and preferably which is alsouseful in commercial bioreactor applications.

1.2.6 Segregational Plasmid Maintenance Functions

Stable lower copy number plasmids typically employ a partitioningfunction that actively distributes plasmid copies between daughtercells. Exemplary partitioning functions include, without limitation,systems of pSC101, the F factor, the P1 prophage, and IncFII drugresistance plasmids. Such functions are referred to herein as “SEG”functions

1.2.7 Post-Segregational Killing (PSK) Functions

Naturally occurring PSK plasmid maintenance functions typically employ atwo component toxin-antitoxin system and generally operate as follows:The plasmid encodes both a toxin and an antitoxin. The antitoxins areless stable than the toxins, which tend to be quite stable. In aplasmidless daughter cell, the toxins and anti-toxins are no longerbeing produced; however, the less stable antitoxins quickly degrade,thereby freeing the toxin to kill the cell.

The toxins are generally small proteins and the antitoxins are eithersmall proteins (proteic systems such as phd-doc) or antisense RNAs whichbind to the toxin-encoding mRNAs preventing their synthesis (antisensesystems such as hok-sok).

Balanced lethal systems discussed below in Section 1.2.7.3 are anexample of an artificial PSK function.

1.2.7.1 Proteic Maintenance System: The phd-doc System

In proteic PSK functions, both the toxin and antitoxin are synthesizedfrom operons in which the gene encoding the antitoxin is upstream of thegene encoding the toxin. These operons autoregulate transcriptionlevels, and synthesis of the encoded proteins is translationallycoupled. The antitoxin is generally synthesized in excess to ensure thattoxin action is blocked. The unstable antitoxins are constantly degradedby host-encoded proteases, requiring constant synthesis of antitoxin toprotect the cell. Upon loss of the plasmid, antitoxins are no longerproduced, and the existing antitoxins rapidly degrade, permitting thetoxin to kill the host cell.

The phd-doc system is an example of a proteic PSK function. The phd-docsystem occurs naturally within the temperate bacteriophage P1, whichlysogenizes Escherichia coli, as an ˜100 kb plasmid. This maintenancelocus encodes two small proteins: the toxic 126 amino acid Doc proteincauses death on curing of the plasmid by an unknown mechanism, and the73 amino acid Phd antitoxin prevents host death, presumably by bindingto and blocking the action of Doc.

Phd and Doc are encoded by a single transcript in which the ATG startcodon of the downstream doc gene overlaps by one base the TGA stop codonof the upstream phd gene. Expression of these two proteins is thereforetranslationally coupled, with Phd synthesis exceeding synthesis of thetoxic Doc protein.

In addition, transcription of this operon is autoregulated at the levelof transcription through the binding of a Phd-Doc protein complex to asite which blocks access of RNA polymerase to the promoter of the operonas concentrations of both proteins reach a critical level. Although Docappears to be relatively resistant to proteolytic attack, Phd is highlysusceptible to cleavage. The PSK mechanism of a plasmid-encodedphd-doclocus is therefore activated when bacteria spontaneously lose thisresident plasmid, leading to degradation of the Phd antitoxin andsubsequent activation of the Doc toxin which causes cell death.

1.2.7.2 Antisense Maintenance System: The hok-sok System

In antisense maintenance systems, the antitoxins are antisense RNAs thatinhibit translation of toxin-encoding mRNAs. Like the antitoxinpeptides, the antisense RNAs are less stable than the toxin-encodingmRNA. Loss of the plasmid permits existing antitoxins to degrade,thereby permitting synthesis of the toxin which kills the host cell.

An example of an antisense maintenance system is the hok-sok system,encoded by the parB locus of plasmid R1. The system is comprised ofthree genes: hok, sok and mok.

Hok is a membrane-associated protein which irreversibly damages the cellmembrane, killing host cells. Expression of Hok from hok mRNA leads to aloss of cell membrane potential, arrest of respiration, changes in cellmorphology, and cell death.

The sok gene encodes a trans-acting RNA which blocks translation of hokmRNA, thereby preventing Hok killing of host cells. The sok RNA is lessstable than hok mRNA and is expressed from a relatively weak promoter.(Gerdes et al. Annu. Rev. Genet, 31:1–31, 1997) incorporated herein. Themechanism by which sok RNA blocks translation of Hok inplasmid-containing cells became apparent only after the identificationof mok (modulation of killing), a third gene in the parB locus. The mokopen reading frame overlaps with hok, and is necessary for expressionand regulation of hok translation.

The sok antisense RNA forms a duplex with the 5′ end of the mok-hokmessage rendering the mok ribosome binding site inaccessible toribosomes and promoting RNase III cleavage and degradation of the mRNA.In the absence of mok translation, hok is not expressed from intactmessage, even though its own ribosome binding site is not directlyobscured by sok RNA.

When a plasmid-free cell is formed, the unstable sok RNA decays muchmore rapidly than the stable mok-hok message. When the protectionafforded by sok is lost, Mok and Hok are translated and the cell dies.

A limitation of the hok-sok system is that a significant number ofplasmidless cells can arise when the hok-sok system is inactivated bymutations within the Hok open reading frame.

1.2.7.3 Balanced Lethal Systems

In a balanced-lethal system (a PSK function), a chromosomal geneencoding an essential structural protein or enzyme is deleted from thebacterial chromosome or is mutated such that the gene can no longeroperate. The removed or damaged gene is then replaced by a plasmidcomprising a fully operating gene. Loss of the plasmid results in aninsufficiency of the essential protein and the death of the plasmidlesscell.

A balanced-lethal system has been successfully employed in S.typhimurium based on expression of the asd gene encoding aspartateβ.-semialdehyde dehydrogenase (Asd). Asd is a critical enzyme involvedin the synthesis of L-aspartic-.β-semialdehyde, which is a precursoressential for the synthesis of the amino acids L-threonine (andL-isoleucine), L-methionine, and L-lysine, as well as diaminopimelicacid, a key structural component essential to the formation of the cellwall in Gram-negative bacteria. Loss of plasmids encoding Asd would belethal for any bacterium incapable of synthesizing Asd from thechromosome, and would result in lysis of the bacterium due to aninability to correctly assemble the peptidoglycan layer of its cellwall.

The asd system (a PSK function) has been successfully employed inattenuated S. typhimurium-based live vector strains for immunization ofmice with a variety of procaryotic and eucaryotic antigens, includingsuch diverse antigens as detoxified tetanus toxin fragment C and the LTenterotoxin, synthetic hepatitis B viral peptides, and gamete-specificantigens such as the human sperm antigen SP10.

Murine mucosal immunization with these live vector strains has elicitedsignificant immune responses involving serum IgG and secretory IgAresponses at mucosal surfaces.

The asd system has recently been introduced into attenuated Salmonellatyphi vaccine strains in an attempt to increase the stability ofplasmids expressing synthetic hepatitis B viral peptides. However, whenvolunteers were immunized with these live vector strains, no immuneresponse to the foreign antigen was detected.

In fact, to date, very few reports have documented an immune response toplasmid-based expression of a foreign antigen from stabilized plasmidsafter human vaccination with an attenuated S. typhi live vector. In onereport, the vaccine strain Ty21 a was made auxotrophic for thymine byselecting in the presence of trimethoprim for an undefined mutation inthe thyA gene, encoding thymidylate synthetase.

Although in some cases failure of live vector strains may have resultedfrom over-attenuation of the strain itself, it appears probable thatcurrent killing systems for plasmids suffer from additional limitations.In those situations where the chromosomal copy of the gene has beeninactivated, rather than removed, may allow for restoration of thechromosomal copy via homologous recombination with the plasmid-bornegene copy if the bacterial strain utilized is recombination-proficient.

Balanced-lethal systems based on catalytic enzyme production are subjectto a number of important deficiencies. In particular, sincecomplementation of the chromosomal gene deletion requires only a singlegene copy, it is inherently difficult to maintain more than a few copiesof an expression plasmid. The plasmidless host strain must be grown onspecial media to chemically complement the existing metabolicdeficiency.

Moreover, plasmidless cells may also benefit from “cross-feeding”effects when a diffusible growth factor is growth limiting.

There is therefore a need in the art for a Plasmid Maintenance Systemwhich is not solely reliant on a balanced lethal system, particularlyfor use in bacterial live vector vaccines.

2. SUMMARY OF THE INVENTION

The present invention relates generally to a stabilized expressionplasmid comprising a Plasmid Maintenance System and a nucleotidesequence encoding a protein or peptide, such as a foreign antigen, andmethods for making and using such stabilized expression plasmids. ThePlasmid Maintenance System of the present optimizes viability by usingstabilized lower copy number expression plasmids capable of expressinghigh levels of heterologous antigen in response to an environmentalsignal likely to be encountered in vivo after the vaccine organisms havereached an appropriate ecological niche.

In a particular aspect, the stabilized expression plasmid is employed ina Salmonella typhi live vector vaccine, such as the strain CVD908-htrA.

The invention optimizes the maintenance of expression plasmids at twoindependent levels by: (1) removing sole dependence on balanced lethalmaintenance systems; and (2) incorporating a plasmid partition system toprevent random segregation of expression plasmids, thereby enhancingtheir inheritance and stability. In one aspect of the invention, thestabilized expression plasmid is recombinantly engineered to express oneor more antigens, preferably one or more Shiga toxin 2 (Stx2) antigensor substantial homologues thereof, such as Shiga toxin subunit pentamersor a genetically detoxified Stx 2.

The stabilized expression plasmid preferably comprises one or morenon-catalytic plasmid maintenance functions.

In another aspect, the expression plasmid comprises a PlasmidMaintenance System which comprises at least one PSK function and atleast one SEG function. For example, the Plasmid Maintenance System maycomprise a two-component Plasmid Maintenance System comprising one PSKfunction and one SEG function. Alternatively, the Plasmid MaintenanceSystem may comprise a three-component Plasmid Maintenance Systemcomprising a PSK function, a SEG function and another PSK. In apreferred alternative, the Plasmid Maintenance System compriseshok-sok+par+parA+phd-doc; wherein any of the stated functions may bereplaced by a substantial homologue thereof.

The Plasmid Maintenance Systems can be incorporated into multicopyexpression plasmids encoding one or more proteins or peptides ofinterest. Such multicopy expression plasmids produce a gene dosageeffect which enhances the level of expression of the protein or peptideof interest. Where the Plasmid Maintenance System is to be employed in abacterial live vector vaccine, the protein or peptide of interest is oneor more foreign antigens.

In one aspect, the expression plasmid is a vaccine expression plasmidcomprising a Plasmid Maintenance System and at least one antigen, forexample, at least one Shiga toxin 2 (Stx2) antigen and/or substantialhomologue thereof. Where the antigen is a Shiga toxin 2 antigen, theShiga toxin 2 antigen can, for example, be either a B subunit pentameror a genetically detoxified Stx 2.

In another aspect the expression plasmid comprises a Plasmid MaintenanceSystem which incorporates the ssb balanced lethal system and the ssblocus of the bacterial live vector has been inactivated using a suicidevector comprising a temperature sensitive origin of replication. In oneaspect, the bacterial live vector is S. typhi and the suicide vector isused to inactivate the ssb locus of S. typhi. In one aspect, the suicidevector is a derivative of pSC101 which carries sacB, described herein.

In another aspect, the present invention provides a Plasmid MaintenanceSystem incorporating a PSK function involving a silent plasmid addictionsystem based on antisense RNA control mechanisms that only synthesizelethal proteins after plasmid loss has occurred.

In one aspect the expression plasmid comprises a series of expressionplasmids, each comprising self-contained genetic cassettes encodingregulated expression of a heterologous antigen, an origin ofreplication, and a selectable marker for recovering the plasmid.

In one aspect the expression plasmid comprises a Plasmid MaintenanceSystem which incorporates a PSK function based on the ssb gene. In arelated aspect, mutated alleles such as ssb-1, described herein, areincorporated into the expression plasmids to enhance higher copy numberplasmids by over-expression of SSB1-like proteins to form the requiredbiologically active tetramers of SSB.

In another aspect, the expression plasmid comprises a promoter. Thepromoter is preferably an inducible promoter, such as the ompC promoter.In one aspect, the inducible promoter is the mutated P_(ompC1), or theP_(ompC3) promoter described herein.

In one aspect, the expression plasmid of the present invention comprisesa plasmid inheritance (or partition) locus; an origin of replicationselected to provide copy number which effectively stabilizes a givenantigen; a PSK function; and a nucleotide sequence encoding an antigenand a promoter which ultimately controls translation of the antigen andhas a strength which is selected to improve antigen production withoutkilling the cell.

The present invention also provides a method of using the expressionplasmid comprising transforming a bacterial cell using said expressionplasmid, and culturing the bacterial cell to produce the protein orpeptide (e.g., the antigen), and/or administering said transformed cellor cell culture to a subject. Where the transformed bacterial cells areadministered to a subject, they are administered in an amount necessaryto elicit an immune response which confers immunity to the subject forthe protein or peptide. The subject is preferably a human, but may alsobe another animal, such as a dog, horse, or chicken.

In one aspect, an expression plasmid is provided which comprises atleast 3 independently functioning expression cassettes wherein onecassette encodes a protein or peptide of interest and the remainingcassettes each encode a different Plasmid Maintenance Function.

In one aspect, an expression plasmid is provided which encodes (1) atest antigen operably linked to a promoter and (2) a Plasmid MaintenanceSystem.

In another aspect, a regulated test antigen expression cassette isprovided which operates such that as induction of antigen expression isincreased, a metabolic burden is placed on the bacterium which leadsphenotypically to plasmid instability, i.e. a selective advantage iscreated for all bacteria which can spontaneously lose the offendingplasmid. The test antigen can be the green fluorescent protein (GFPuv).The expression cassette encoding the test antigen can also comprise aninducible promoter, such as the ompC promoter, positioned such that theinducible promoter ultimately drives the translation of the testantigen.

In one aspect, a method of making an expression plasmid is providedwhich comprises synthesizing an expression plasmid comprising at least 3independently functioning expression cassettes wherein one cassetteencodes a protein or peptide of interest and the remaining cassetteseach encode a different Plasmid Maintenance Function.

In one aspect, a method of screening Plasmid Maintenance Systems isprovided comprising: providing one expression cassette which encodes aprotein or peptide of interest, and at least two other expressioncassettes, each encoding and capable of expressing in the host bacteriallive vector a different Plasmid Maintenance Function; inserting thethree expression cassettes into a single expression plasmid;transforming a bacterial live vector with the single expression plasmid;culturing the transformed bacterial live vector; and determining therate of introduction of plasmidless cells into the culture.

In one aspect, the present invention comprises an attenuated bacteriallive vector vaccine comprising an attenuated bacterial live vector whichhas been transformed with a stabilized expression plasmid comprising aPlasmid Maintenance System, preferably a non-catalytic plasmidmaintenance system.

In one aspect, the present invention comprises an attenuated bacteriallive vector vaccine comprising an attenuated bacterial live vector whichhas been transformed with an expression plasmid comprising a PlasmidMaintenance System which incorporates at least one PSK system and atleast one SEG system. The attenuated bacterial live vector can, forexample, be S. typhi CVD908-htrA.

The present invention also provides a method for vaccinating a subjectcomprising administering to the subject an amount of a bacterial livevector vaccine sufficient to elicit an enhanced immune response. Thepresent invention also provides a method for preventing a disease byvaccinating a subject using an amount of such bacterial live vectorsufficient to elicit a protective immune response to one or morepathogens of such disease. The subject is preferably a human but mayalso be another animal, such as a horse, cow or pig. For example, thepresent invention provides a method for preventing hemolytic uremicsyndrome (HUS) caused by Shiga toxin 2-producing enterohemorrhagicEscherichia coli by administering to a subject an amount of a bacteriallive vector transformed with a stabilized plasmid encoding at least oneShiga toxin 2 antigen.

In another aspect, the present invention provides a method for screeningPlasmid Maintenance Systems for efficacy, the method comprising:providing expression plasmids comprising the Plasmid Maintenance Systemsdescribed herein and encoding for a protein or peptide of interest, saidexpression plasmids having copy numbers which vary from low copy number(e.g. ˜5 copies per cell) to medium copy number (e.g. ˜15 copies percell) to high copy number (e.g. ˜60 copies per cell); transformingbacterial live vectors with such expression plasmids; and testing forrate of introduction of plasmidless cells and/or rate of growth ofplasmid-containing cells. The modified origins of replication may beorigins of replication from the plasmids pSC101 (low copy number),pACYC184 (medium copy number), and pAT153 (high copy number).Independently functioning plasmid replication cassettes can be utilizedwhich permit testing of the efficiency of one or more plasmidstabilization systems as copy number is increased.

In another aspect, the present invention provides stabilized expressionplasmids for use in attenuated S. typhi live vectors which contain aselectable marker which can readily be replaced by a non-drug resistantlocus or by a gene encoding an acceptable drug resistance marker such asaph encoding resistance to the aminoglycosides kanamycin and neomycin.

The Plasmid Maintenance Systems of the present invention provideimproved stability of recombinant plasmids, overcoming prior artproblems of plasmid instability, for example, in bioreactor and livevector vaccination uses. The plasmids of the present invention arespecifically tailored for vaccine applications though such plasmids arealso useful in large scale protein production.

The plasmids of the present invention are a major improvement over theprior art in that they overcome the problems associated with plasmidlesstakeover and plasmid instability and have wide ranging utility in fieldssuch as commercial protein production and attenuated bacterial livevector vaccine production.

There has long been a need for a solution to the problems of plasmidlesstakeover and plasmid stability associated with the field of vaccinedelivery and protein production. The present invention solves this longfelt need.

3. Definitions

The term “Plasmid Maintenance System” (“PMS”) as used herein refers to anucleotide sequence comprising at least one post-segregational killingfunction (“PSK”) and at least one partitioning or segregating system(“SEG”), and optionally including any other Plasmid MaintenanceFunction.

The term “Plasmid Maintenance Function” is used herein to refer to anyplasmid-stability enhancing function associated with a PMS. The termincludes both naturally-occuring nucleotide sequences encoding plasmidmaintenance functions, as well as nucleotide sequences which aresubstantially homologous to such naturally-occurring plasmid maintenancefunctions and which retain the function exhibited by the correspondingnaturally-occurring plasmid maintenance function.

The term “Post-Segregational Killing System” (PSK) is used herein torefer to any function which results in the death of any newly dividedbacterial cell which does not inherit the plasmid of interest, andspecifically includes balanced-lethal systems such as asd or ssb,proteic systems such as phd-doc, and antisense systems such as hok-sok.The term includes both naturally-occuring nucleotide sequences encodingsuch PSKs, as well as nucleotide sequences which are substantiallyhomologous to such naturally-occurring nucleotide sequences and whichretain the function exhibited by the corresponding naturally-occurringnucleotide sequences.

The term “substantially homologous” or “substantial homologue,” inreference to a nucleotide sequence or amino acid sequence, indicatesthat the nucleic acid sequence has sufficient homology as compared to areference sequence (e.g., a native sequence) to permit the sequence toperform the same basic function as the corresponding reference sequence;a substantially homologous sequence is typically at least about 70percent sequentially identical as compared to the reference sequence,typically at least about 85 percent sequentially identical, preferablyat least about 95 percent sequentially identical, and most preferablyabout 96, 97, 98 or 99 percent sequentially identical, as compared tothe reference sequence. It will be appreciated that throughout thespecification, where reference is made to specific nucleotide sequencesand/or amino acid sequences, that such nucleotide sequences and/or aminoacid sequences may be replaced by substantially homologous sequences.

The terms “Segregating System” and/or “Partitioning System” (bothreferred to herein as “SEG”) are used interchangeably herein to refer toany plasmid stability-enhancing function that operates to increase thefrequency of successful delivery of a plasmid to each newly dividedbacterial cell, as compared to the frequency of delivery of acorresponding plasmid without such a SEG system. SEG systems include,for example, equipartitioning systems, pair-site partitioning systems,and the par locus of pSC101. The term includes both naturally-occuringnucleotide sequences encoding such SEG systems, as well as nucleotidesequences which are substantially homologous to such naturally-occurringnucleotide sequences and which retain the function exhibited by thecorresponding naturally-occurring nucleotide sequences.

The term “detoxified” is used herein to describe a toxin having one ormore point mutations which significantly reduce the toxicity of thetoxin as compared to a corresponding toxin without such point mutations.

The term “immunizingly effective” is used herein to refer to an immuneresponse which confers immunological cellular memory upon the subject,with the effect that a secondary response (to the same or a similartoxin) is characterized by one or more of the following characteristics:shorter lag phase in comparison to the lag phase resulting from acorresponding exposure in the absence of immunization; production ofantibody which continues for a longer period than production of antibodyfor a corresponding exposure in the absence of such immunization; achange in the type and quality of antibody produced in comparison to thetype and quality of antibody produced from such an exposure in theabsence of immunization; a shift in class response, with IgG antibodiesappearing in higher concentrations and with greater persistence thanIgM; an increased average affinity (binding constant) of the antibodiesfor the antigen in comparison with the average affinity of antibodiesfor the antigen from such an exposure in the absence of immunization;and/or other characteristics known in the art to characterize asecondary immune response.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1C: Genetic maps of exemplary pGEN expression plasmids (pGEN2,pGEN3, and pGEN4) of the present invention.

FIGS. 2A–2D: Genetic maps of exemplary oriE -based expression plasmids(pJN72, pJN51, pJN10, and pJN12) of the present invention.

FIGS. 3A–H: Flow cytometry histograms of GFP fluorescence for CVD908-htrA carrying expression vectors with the hok-sok post-segregationalkilling system.

FIGS. 4A–D: Complete pGEN2 nucleotide sequence (SEQ ID NO:1), comprisingnucleotides 1–4196.

FIGS. 5A–B: Partial pGEN3 nucleotide sequence (SEQ ID NO:2), comprisingnucleotides 1201–2397 and showing the sequence of ori15A.

FIGS. 6A–C: Partial pGEN4 nucleotide sequence (SEQ ID NO: 3), comprisingnucleotides 1201–3848 and showing the sequence of ori101.

FIGS. 7A–7E: Genetic maps of exemplary ori15A–based pGEN expressionplasmids (pGEN91, pGEN111, pGEN121, pGEN193, and pGEN222) of the presentinvention.

FIGS. 8A–C: Flow cytometry histograms of GFP fluorescence for expressionplasmids pGEN91, pGEN111, pGEN121, pGEN193, and pGEN222.

5. DETAILED DESCRIPTION OF THE INVENTION

Bacterial live vector vaccines employ a bacterial live vector to expressgenes encoding protective antigens of bacterial, viral or parasiticpathogens. The bacterial protective antigens are preferably non-nativeto the bacterial live vector, i.e. heterologous. The bacterial livevector vaccine is administered to a host, thereby exposing the expressedantigens to the host's immune system, eliciting an immune response ofappropriate character to confer immunity to the host.

In order to achieve enhanced immunogenicity, the plasmids expressingsuch protective antigens must be stabilized. To the inventor'sknowledge, no currently available S. typhi-based Plasmid MaintenanceSystem takes advantage of naturally occurring partition mechanisms knownto improve the stability of multicopy plasmids in other strains.

The present invention provides a non-catalytic Plasmid MaintenanceSystem for the stabilization of expression plasmids encoding foreignantigens in a S. typhi live vector vaccine strain. In one aspect the S.typhi strain is CVD 908-htrA. In another aspect, the present inventionimproves and/or optimizes maintenance of expression plasmids byproviding Plasmid Maintenance Systems which operate at two independentlevels: (1) removing sole dependence on catalytic balanced lethalmaintenance systems; and (2) incorporating a plasmid partition systemwhich will prevent random segregation of the expression plasmids,thereby enhancing their inheritance and stability. A critical reason forpursuing this particular approach is that this method of improvingplasmid maintenance involves no additional manipulations of the livevector strain, and therefore can improve the immunogenicity ofheterologous antigens expressed within any live vector strain.

The non-catalytic Plasmid Maintenance System of the present inventionimproves the stability of multicopy expression plasmids within abacterial live vector vaccine, such as CVD908-htrA.

In one aspect, the present invention incorporates the naturallyoccurring PSK function hok-sok from the antibiotic-resistance factorpR1, or a substantial homologue thereof, within multicopy expressionplasmids. The hok-sok system is a silent plasmid addiction system basedon antisense RNA control mechanisms that only results in synthesis oflethal proteins after plasmid loss has occurred.

The present invention also provides a plasmid maintenance systemcomprising a complementation-based PSK function in which the chromosomalgene ssb, encoding the essential non-catalytic single-stranded bindingprotein (SSB) required for DNA replication, is specifically deleted andinserted within a multicopy expression plasmid.

The present invention also provides an improved Plasmid MaintenanceSystem comprising an expression plasmid encoding at least one SEG locusand at least one PSK function.

5.1 Suicide Vectors

Heterologous antigens can be expressed within live vector strains, suchas CVD908-htrA, from genes residing either on plasmids or integratedwithin the chromosome. One technique for integrating these genes intothe host chromosome involves the use of temperature sensitive “suicidevectors” such as pIB307 which contains a temperature-sensitive origin ofreplication from pSC101 (ori101^(ts)). The present invention provides animproved suicide vector for use in CVD908 and CVD908-htrA, derived frompIB307 which allows for easier construction of mutagenesis cassettes toalter the live vector chromosome.

Integration of these suicide vectors into the chromosome by homologousrecombination results from temperature inactivation of the plasmidreplication protein, RepA, a protein essential to the function ofori101. Spontaneous resolution of the resulting unstable merodiploidintermediates is detected by counter-selection for loss of the sacB genecontained on the resolving suicide vector. The sacB gene contained onall excised plasmids encodes the levansucrase enzyme, which is lethalwhen expressed within the cytoplasm of enteric bacteria, including S.typhi, growing in the presence of sucrose. Since resolving merodiploidsare selected by incubating in the presence of 10% sucrose, excisedplasmids will kill host bacteria unless they cure spontaneously.

This system was successfully used to integrate a kanamycin-resistancecassette into the ΔaroC1019 locus of CVD908. However, these experimentswere successful because the gene being mobilized into the chromosome ofS. typhi encoded a selectable drug-resistance marker. Using these earlyvectors, replacing the kanamycin-resistance cassette with anon-selectable marker was not successful because, although the incomingmarker could be integrated into the chromosome as a merodiploid,resolution of the merodiploid to replace the drug resistance gene wasnever detected.

The present invention also provides a method for using such suicidevectors to inactivate the ssb locus of attenuated Salmonella typhistrains such as CVD908-htrA.

The present invention allows such suicide vectors to permit efficientmobilization of genes expressing proteins or peptides of interest, suchas heterologous antigens, into the chromosome of S. typhi CVD908-htrA intwo stages. For example, the present inventor introduced a sacB-aphcassette into the ΔaroC1019 locus, which was then selected usingkanamycin. Generation of this S. typhi CVD908-htrAΔaroC1019::sacB-aphstrain produced a valuable intermediate strain into which, in theory,any structural gene can be efficiently inserted into the aroC locus bymarker-exchange. The sacB gene is used as a counter-selectable marker bypassing merodiploids in the presence of 10% sucrose to select forreplacement of the sacB-aph cassette with the incoming antigen cassette,since resolution of merodiploids in the presence of sucrose will resultin loss of the sacB gene, in order to produce viable progeny. Thisintermediate strain was employed to efficiently integrate thenon-toxigenic mutant LT-K63 of the E. coli heat-labile enterotoxin,creating CVD908ΔaroC1019::LT-K63.

5.2 Plasmid-Based Expression of Heterologous Antigens

Although chromosomal integration of foreign genes confers stability tosuch sequences, the genetic manipulations involved can be difficult, andthe drop in copy number of the heterologous gene often results inproduction of insufficient levels of heterologous antigen to ensure anoptimal immune response.

In contrast, plasmid stability is a complex phenomenon which depends onmultiple factors including (1) copy number of the plasmid; (2)appropriately regulated expression of genes contained within theplasmid; and (3) selective pressure for ensuring the proper segregationand inheritance of the plasmid.

To ensure stability plasmids must be replicated in a regulated manner toprevent their copy number from rising to lethal levels.

In addition, plasmids must segregate during the division of a growingbacterium to ensure that each daughter cell receives at least one copyof the plasmid. Segregation can be a passive, random event or an activeprocess involving synthesis of novel proteins which aid in plasmidsegregation and inheritance. Successful inheritance of randomlysegregating plasmids relies on a high enough copy number of randomlydistributed plasmids within a dividing bacterium to virtually guaranteeinheritance of at least one plasmid by each daughter cell.

The commonly used plasmid cloning vectors, including medium copy numberpBR322 derivatives and high copy number pUC plasmids, are inherited byrandom segregation.

Active segregation involves the synthesis of proteins which are proposedto bind to such plasmids and further coordinate with the membranes ofdividing bacteria to ensure that each daughter receives at least oneplasmid copy. Plasmids employing such active partitioning systems aretypically very low copy number plasmids such as the F sex factor of E.coli or antibiotic resistance R-factors such as pR1 and pRK2.

The present invention exploits naturally occurring SEG functions toenhance inheritance of multicopy expression plasmids, which wouldotherwise be inherited by random segregation, to increase the stabilityof these plasmids.

The present invention also takes advantage of other naturally occurringgenetic systems in which daughter cells which do not successfullyinherit an expression plasmid will be killed and removed from thegrowing population, i.e., PSK functions. The incorporation of more thanone category of plasmid stabilization function is referred to herein asa Plasmid Maintenance System. For example, the incorporation of both aSEG function such as a partition locus and a PSK function into a singleexpression plasmid yields a Plasmid Maintenance System.

It should be noted that a gene conferring resistance to a bactericidalantibiotic, such as the aph gene encoding resistance to kanamycin andneomycin, is also considered a PSK function, as is the asd-basedbalanced-lethal system.

5.3 Balanced Lethal Systems

One method of ensuring the inheritance of expression plasmids involvesthe construction of a PSK system or a substantial homologue thereof,referred to as a balanced lethal system, for plasmids expressingheterologous antigens. In a plasmid-based balanced lethal system,plasmids replicating in the cytoplasm of the bacterium express acritical protein required by the bacterium to grow and replicate. Lossof such plasmids removes the ability of the bacterium to express thecritical protein and results in cell death.

The asd system has recently been introduced into attenuated S. typhivaccine strains in an attempt to increase the stability of plasmidsexpressing synthetic hepatitis B viral peptides.

However, when volunteers were immunized with these live vector strains,no immune response to the foreign antigen was detected. See Tacket etal., Infection and Immunity, 65:3381, 1997 (incorporated herein byreference). In fact, to date, few reports have documented an immuneresponse to plasmid-based expression of a foreign antigen from plasmids(stabilized or otherwise) after vaccination of humans with an attenuatedS. typhi live vector.

Although in some cases failure of live vector strains may have resultedfrom over-attenuation of the strain itself, the inventor's conclusion isthat currently used PSK functions for plasmids suffer from additionallimitations, in particular, from segregation limitations and catalyticactivity limitations. The present invention provides improved expressionplasmids comprising enhanced segregation capabilities by incorporatingat least one partitioning system along with at least one PSK system.

5.4 Segregation Limitations

One limitation of plasmid maintenance functions such as the asd function(as well as the thyA function) is that they do not enhance theinheritance of resident plasmids, which continue to segregate randomlywith or without the presence of the asd function. Therefore, if residentexpression plasmids carrying asd genes are inherently unstable, theywill be lost, regardless of the requirement of the bacterium for Asd.

The inherent stability of an asd expression plasmid can be defined bygrowing plasmid-bearing strains in the presence of DAP, which removesthe selective pressure that ensures that all viable bacteria contain theexpression plasmid. If a given plasmid is inherently unstable, it willbe lost from bacteria at a high rate and such plasmidless bacteria willlyse in the absence of growth supplements; the overall result of thiseffect will be a population of bacteria that grows much slower thanwildtype unaltered strains.

The present invention improves plasmid stability by incorporating a SEGfunction, such as a partition locus, or a substantial homologue of a SEGfunction, onto the expression plasmid to enhance the inheritance of suchplasmids by actively dividing bacteria. Partition loci are naturallypresent on the virulence plasmids of S. typhimurium. Tinge and Curtiss,Journal of Bacteriology, 172:5266, 1990 (incorporated herein byreference) reported that such partition loci were well conserved amongS. typhimurium virulence plasmids, and that when a 3.9 kb restrictionfragment encoding this locus was introduced onto the lower copy numberplasmid pACYC184 (˜15 copies per cell), the observed plasmid stabilityincreased from 34% plasmid-containing cells to 99% plasmid-bearing cellsafter 50 generations. The nucleotide sequence of this locus was laterdetermined by Cerin and Hackett, Plasmid, 30:30, 1993 (incorporatedherein by reference), (GenBank Accession Number M97752).

5.5 Catalytic Activity Limitations

Another potential limitation of a plasmid maintenance function such asthe asd function (as well as the thyA system) is its reliance on anenzyme with catalytic activity. Given that complementation with only asingle copy of the asd gene is sufficient to remove auxotrophy, it isnot clear why all copies of a multicopy plasmid should remain stable,especially if they encode an especially problematic heterologous antigenwhich inhibits growth of the bacterium.

Further, although higher copy number expression plasmids may expressappreciable levels of a given heterologous antigen in vitro, suchplasmids may not be maintained at the expected copy numbers in vivo dueto toxicity and may in fact be present at much lower copy numbers, whichwould be expected to reduce any observed immune response specific forthe heterologous antigen. Accordingly, the present invention thusprovides stably maintained low and medium copy number plasmids forexpressing heterologous antigens.

5.6 The Non-Catalytic ssb PSK Function

The potential limitation of catalytic activity associated with balancedlethal systems is addressed here through the use of plasmids expressingthe single-stranded binding protein (SSB) from S. typhi totrans-complement an otherwise lethal mutation introduced into thechromosomal ssb gene. The biochemistry and metabolic roles of the E.coli SSB protein have been extensively reviewed in Lohman et al., AnnualReviews in Biochemistry 63:527, 1994 and Chase et al., Annual Reviews inBiochemistry 55:103, 1986 (the disclosures of which are incorporatedherein by reference).

SSB is a non-catalytic 177 amino acid protein, with a relative molecularweight of 19 kDa, that binds with high affinity to single-stranded DNA(ssDNA), and plays an essential role as an accessory protein in DNAreplication, recombination, and repair. The biologically relevant formof SSB involved in binding to ssDNA is a tetramer, which binds in twomodes to ssDNA, intimately associating with an average of either 35(SSB₃₅-binding mode) or 65 bases (SSB₆₅-binding mode). The specificconditions controlling the preferred mode of binding are complex anddepend on the surrounding concentration of monovalent and divalentsalts, pH, and temperature, as well as the amount of SSB proteinpresent. Under given conditions, high concentrations of SSB favor theSSB₃₅-binding mode, with lower SSB concentrations favoring theSSB₆₅-mode. However, it must be emphasized that in both binding modes,the required conformation of SSB is a tetramer.

Spontaneously occurring temperature-sensitive point mutations within thessb gene have now been characterized at the biochemical, physiological,and nucleotide level; one such mutant, ssb-1, contains the pointmutation His 55 to Tyr, and has been found to be unable to assemblecorrectly into tetramers at non-permissive temperatures and naturalexpression levels. These mutant strains exhibit temperature-sensitivelethal defects in DNA replication and recombination.

The segregation frequencies of plasmids carrying ssb which complementchromosomal ssb mutations in E. coli bacteria were examined by Porter etal. Bio/Technology 8:47, 1990 (incorporated herein by reference). Theyobserved that in experiments involving bioreactors, the segregationfrequency in plasmid-bearing strains growing in continuous culture undernon-selective conditions for 150 hours was less than 1×10⁻⁷; thissegregation frequency was independent of copy number, as both lower copynumber pACYC184 plasmids and very high copy number pUC19 plasmids weremaintained at the same frequency. However, it must be noted that theplasmids involved expressed only a drug-resistance marker in addition tothe SSB protein.

The present invention provides an improved plasmid maintenance systemwhich incorporates a partition locus such as that present on pSC101, ora substantial homologue of such partition locus, and may alsoincorporate an active partitioning system, or a substantial homologuethereof, such as that described above for the virulence plasmid of S.typhimurium.

The present invention removes dependence on catalytic enzymes to conferplasmid stability. In one aspect, mutated alleles similar to ssb-1 areintroduced into the expression plasmids to enhance higher copy numberplasmids by overexpression of SSB1-like proteins to form the requiredbiologically active tetramers of SSB. In another aspect the presentinvention provides a PSK function involving a silent plasmid addictionsystem based on antisense RNA control mechanisms that only synthesizelethal proteins after plasmid loss has occurred.

5.7 Expression Plasmids and Self-Contained Genetic Cassettes

The present invention also comprises a series of expression plasmidswhich are referred to herein as pGEN plasmids. pGEN plasmids compriseself-contained genetic cassettes encoding regulated expression of aheterologous antigen, an origin of replication, and a selectable markerfor recovering the plasmid. This vector series has been specificallydesigned to test whether any Plasmid Maintenance System can increase thestability of plasmids, for example within an attenuated S. typhi vaccinebackground.

The basic structure of these vectors is represented in FIG. 1, and thecomposite gene sequence for the vector pGEN2 (SEQ ID NO: 1) isrepresented in FIG. 4; FIGS. 5 & 6 show specific composite sequences forthe origins of replication in pGEN3 and pGEN4 respectively.

It is critical to note that the pGEN plasmids are designed to comprise 3independently functioning genetic cassettes. These cassettes have beenconstructed such that individual components can be optimized byreplacement as necessary. Accordingly, in addition to the variousPlasmid Maintenance Systems described herein, the cassettes can testother promising systems now in existence or which may become availablein the future. Further, the optimized plasmid(s) can be adapted toexpress relevant protective heterologous antigens within attenuatedvaccine strains for immunization of humans.

The pGEN plasmids provide a regulated test antigen expression cassettewhich operates such that as induction of antigen expression isincreased, a metabolic burden is placed on the bacterium which leadsphenotypically to plasmid instability, i.e. a selective advantage iscreated for all bacteria which can spontaneously lose the offendingplasmid. Thus one aspect of the present invention provides aconditionally unstable plasmid which can be examined for stability asplasmid maintenance systems are incorporated.

In a preferred mode, the regulated test antigen expression cassettecontained within the pGEN plasmids comprises the inducible ompCpromoter, or a substantial homologue thereof, driving expression of adetectable protein, such as the codon-optimized green fluorescentprotein (GFPuv, available from Clontech), overexpression of which istoxic to E. coli and S. typhi.

The present invention also comprises a series of plasmid repliconshaving copy numbers which vary from low copy number (i.e., ˜1 to ˜10,preferably ˜5 copies per cell) to medium copy number (i.e., ˜11 to ˜25,preferably ˜15 copies per cell) to high copy number (i.e., ˜26 to ˜100,preferably ˜60 copies per cell). To accomplish this, origins ofreplication from the well-characterized plasmids pSC101, pACYC184, andpAT153 have been modified using polymerase chain reaction (PCR)techniques to create independently functioning plasmid replicationcassettes. These replication cassettes permit testing of the efficiencyof a plasmid maintenance system as copy number is increased.

The present invention also comprises selectable expression plasmids foruse in attenuated S. typhi live vectors. These expression plasmidscontain a selectable marker which can ultimately be replaced either by anon-drug resistant locus, such as ssb, or by a gene encoding anacceptable drug resistance marker such as aph encoding resistance to theaminoglycosides kanamycin and neomycin.

To accomplish this, resistance cassettes encoding resistance tocarbenicillin and tetracycline have been constructed, with transcriptionbeing efficiently terminated by an rrnB T1T2 terminator. A detaileddescription of the individual components comprising the expression andreplication cassettes follows.

Specific components of the Plasmid Maintenance System can besystematically inserted into the basic expression replicons to assessany individual or synergistic influence of these functions on plasmidstability in the presence and absence of selection. For example, apost-segregational killing function (e.g., the hok-sok locus) can beinserted as an EcoRI-XbaI cassette, such that flanking transcriptionfrom surrounding loci, such as the antigen and selection cassettes, isdivergent and will not significantly disturb the wild type transcriptionlevels which control the lethality of this locus (FIG. 7B, PGEN111).

Similarly, the par passive partition locus can be inserted as aBamHI-BglII fragment between the origin of replication and selectioncassettes (FIG. 7C, pGEN 121). Interestingly, in the work leading to thepresent invention, it was observed that the orientation of the par locusenhances synthesis of GFPuv on solid medium when inserted in the naturalorientation found within ori101 of pSC101; this orientation was adoptedfor all of the expression plasmids.

The active partitioning locus is preferably the parA locus, constructedas an XhoI-EcoRI cassette from the same pR1 resistance plasmid fromwhich hok-sok was adapted. To preserve natural transcription levels andregulation within this locus, the cassette is preferably positionedwithin an area of the expression plasmids such that flankingtranscription progresses away from parA (FIGS. 7D and 7E, pGEN193 andpGEN222).

5.8 Components of the Antigen Expression and Replication Cassettes

5.8.1 Promoter

It will be appreciated by one of skill in the art that a wide variety ofcomponents known in the art may be included in the expression cassettesof the present invention, including a wide variety of transcriptionsignals, such as promoters and other sequences that regulate the bindingof RNA polymerase to the promoter. The operation of promoters is wellknown in the art and is described in Doi, Regulation of Gene Expression,Modern Microbial Genetics pages 15–39 (1991) (the entire disclosure ofwhich is incorporated herein by reference). The ensuing description usesthe ompC promoter by way of example, and is not meant to delimit theinvention.

The promoter is preferably an environmentally regulatable promotorcontrolled by a biologically relevant signal such as osmolarity. In apreferred mode, the promoter is the ompC promoter. The ompC gene encodesa porin protein which inserts as a trimer into the outer membrane of abacterial cell. Expression and control of ompC is complex and hasrecently been reviewed in considerable detail in Pratt et al., MolecularMicrobiology 20:911, 1996 and Egger et al., Genes to Cells 2:167, 1997(the disclosures of which are incorporated herein by reference).

Synthesis of the ompC protein is ultimately controlled at the level oftranscription by the osmolarity of the surrounding environment such thatincreases in osmolarity are accompanied by increases in thetranscription of ompC. However, increases in osmolarity do not directlymediate increases in the transcription of ompC. Rather, the bacteriumsenses the surrounding osmolarity using a two-component signaltransduction system encoded by the ompB operon. This operon is composedof two genes transcribed in the order envZ-ompR. The envZ gene encodes a450 amino acid (a.a.) protein, containing two transmembrane regions,which inserts into the bacterial inner membrane (perhaps as a dimer)with an N-terminal 118 a.a. osmotic-sensing domain extending into theperiplasmic space and a C-terminal 270 a.a. catalytic domain extendinginto the cytoplasm. The C-terminal catalytic domain possesses bothkinase and phosphatase activities which are modulated by osmolarity suchthat as osmolarity increases, kinase activity predominates, and asosmolarity drops, phosphatase activity predominates.

EnvZ kinase activity phosphorylates aspartic acid residue 55 of the 239a.a. cytoplasmic protein OmpR, creating OmpR-P. It is the OmpR-Pmodified protein which binds to the ompC promoter and activatestranscription by RNA polymerase; therefore, as osmolarity increases,increasing kinase activity of EnvZ produces higher levels of OmpR-P,which in turn lead to greater transcription of ompC. OmpR-P binds to aregion of the ompC promoter spanning bases −41 (relative to thetranscriptional start site of +1) to −102, with initial binding ofOmpR-P to bases −78 through −102 being followed by additional binding tobases extending to −41 as the concentration of OmpR-P increases withosmolarity. In addition, OmpR-P has been shown to bind to an AT-richupstream region extending back to base −405 which further enhances ompCtranscription.

In a preferred embodiment the ompC promoter fragment from E. coli spansnucleotides +70 through −389. This promoter can direct transcriptionwithin attenuated S. typhi strains of an antibiotic resistance gene,such as the kanamycin resistance gene in an osmotically sensitivemanner. For example, our experiments have demonstrated that when theconcentration of NaCl in liquid growth medium was increased from 0 mM to300 mM, resistance to kanamycin increased from 0 μg/ml to >800 μg/ml.

5.8.2 Origin of Replication

Due to varying degrees of toxicity associated with differentheterologous antigens (i.e. higher toxicity for antigens derived fromparasitic organisms such Plasmodium falciparum vs. virtually no toxicityfor the fragment C of tetanus toxin), the present invention provideslive vector vaccines which preferably express such antigens from eitherlow or medium copy plasmids. It will be appreciated by one skilled inthe art that the selection of an origin of replication will depend onthe degree of toxicity, i.e., the copy number should go down as toxicityto the bacterial strain goes up. In a preferred mode, the PlasmidMaintenance System(s) used are capable of stabilizing replicons of lowor medium copy numbers.

It is preferable for the origin of replication to confer an average copynumber which is between about 2 and about 75. In a preferred mode theorigin of replication is selected to confer an average copy number whichis between about 5 and about 50. More preferably the range is from about5 to about 30. Optimally, the range is from about 15 to about 20.

In one aspect, the origin of replication is from pSC101, conferring acopy number of approximately 5 per genome equivalent.

The oriE1 locus specifies synthesis of a 555 base transcript called RNAI and synthesis of a 110 base antisense RNA transcript called RNA II. AsRNA I is synthesized, the 5′-proximal region of the transcript adopts astem-loop structure composed of 3 domains which can hybridize to acomplementary stem-loop structure formed by RNA II, resulting in adouble stranded RNA–RNA structure forming which causes plasmidreplication to abort.

As synthesis of RNA I continues, generating the full-length 555 basetranscript, a rearrangement of the secondary structure of the transcriptdestroys the initial 3 domain stem-loop structure to form an alternatestem-loop configuration which no longer hybridizes to RNA II. Formationof this alternate structure allows the transcript to hybridize to oneDNA strand of the plasmid itself, forming an RNA-DNA complex which isnicked by endogenous RNAse H to trigger synthesis of the first DNAstrand of the plasmid and plasmid replication.

Plasmid replication is therefore controlled by synthesis of RNA I, whichundergoes a cascade of structural configurations leading to initiationof replication. The necessary progression of the RNA I folding cascade(and resulting replication initiation) is interrupted by competition ofthe domains with RNA II. This mechanism is essentially the same inplasmids containing either oriE1 or ori15A.

The reason these two types of plasmids can coexist within the samebacterium is due to sequence divergence within the region ofhybridization between RNA I and RNA II, such that the RNA II from ori15Awill not hybridize to RNA I from oriE1; this sequence divergence alsoaffects the stability of the RNA I: RNA II hybrid, accounting for thedifferences in copy number between plasmids carrying the oriE1 or ori15Aorigins of replication.

The structural organization of the engineered origins of replicationcassettes for pSC101 (ori101; ˜5 copies per genome equivalent), pACYC184(ori15A derivative; ˜15 copies per genome equivalent), and pAT153 (oriE1derivative; ˜60 copies per genome equivalent) are analogous in structureand function.

5.8.3 Expressed Protein or Peptide

When the expression cassette is used to screen Plasmid MaintenanceSystems, it preferably expresses a protein or peptide with no metabolicactivity. A preferred protein is the green flourescent protein (GFP) ofthe bioluminescent jellyfish Aequorea Victoria, a 238 amino acid proteinwhich undergoes a post-translational modification in which 3 internalamino acids (⁶⁵ Ser-Tyr-Gly⁶⁷) are involved in a cyclization andoxidation reaction. The resulting fluorophore emits blue-green lightmaximally at a wavelength of 509 nm upon irradiation with long-waveultraviolet light at a wavelength of 395 nm. In addition, fluorescenceactivity is remarkably constant over a wide range of pH from 5.5–12 andat temperatures up to 70° C.

Since GFP has no known catalytic activity, the level of observedfluorescence within individual bacteria expressing GFP can provide adirect indication of transcription levels of the gfp gene carried byeach bacterium. Expression of the GFP protein has now been quantitatedin a variety of both prokaryotic and eukaryotic cells and requires noadditional cofactors or enzymes from A. victoria. Fluorophore formationis apparently dependent either on ubiquitous enzymes and cofactors, oris an autocatalytic event.

Individual bacteria expressing GFP can be quantitated either alone orwithin macrophages, epithelial cell lines, and infected animal tissuesusing flow cytometry. GFP fluorescence is absolutely dependent onresidues 2–232 of the undenatured protein. However, fusion of unrelatedbiologically active protein domains to the N-terminus of GFP has stillresulted in fusion proteins with the expected heterologous biologicalactivity which continue to fluoresce as well.

It has been confirmed by sequence analysis (Clontech) that the gfpallele preferred here (i.e. gfpuv) expresses a GFP mutant (GFPuv)containing 3 amino acid substitutions (not involving the fluorophore)which increase fluorescence 18-fold over that of wildtype GFP.

In addition, 5 rarely used arginine codons have been optimized forefficient expression of GFP in E. coli. Since comparison of expressionlevels of various heterologous proteins in E. coli and CVD908 hasdemonstrated equivalent or superior expression within CVD908, it wasexpected that gfpuv will function efficiently in CVD908-htrA.

A coding sequence is inserted in a correct relationship to a promoterwhere the promoter and the coding sequence are so related that thepromoter drives expression of the coding sequence, so that the encodedpeptide or protein is ultimately produced. It will be understood thatthe coding sequence must also be in correct relationship with any otherregulatory sequences which may be present.

5.8.4 Heterologous Antigens

The expression plasmids of the present invention preferably express anantigen for presentation to a host to elicit an immune responseresulting in immunization and protection from disease. While Shigatoxins are presented herein as examples of antigens usefully expressedby the vaccine expression plasmids disclosed herein, the invention isbroad in scope and encompasses the expression of any antigen which doesnot destroy the bacterial live vector and which elicits an immuneresponse when the bacterial live vector containing said expressionplasmid(s) is administered to a host, i.e., a human or other animal.

The vaccine expression plasmids provided herein are used to geneticallytransform attenuated bacterial strains, preferably strains used forhuman vaccination and most preferably used to transform attenuated S.typhi vaccine strains such as CVD908-htrA, and preferably encode eitherthe B subunit of Stx2 or a genetically detoxified Stx2 holotoxin.

A subset of STEC most often referred to as enterohemorrhagic E. coli(EHEC) are capable of causing severe clinical syndromes includinghemorrhagic colitis, hemolytic uremic syndrome (HUS) and thromboticthrombocytopenic purpura (TTP) in a small proportion of infectedindividuals, in addition to causing non-bloody diarrhea in most others.

Hemorrhagic colitis is characterized by copious bloody diarrhea, usuallywithout fever or with only low-grade fever and a relative paucity offecal leukocytes demonstrable in the diarrheal stools. These featuresdifferentiate hemorrhagic colitis from dysentery caused by Shigellawhich is typically scanty stools of blood and mucus, preceded by highfever and with large numbers of fecal leukocytes visible by microscopy.

HUS, a potentially fatal disease that most often affects young childrenbut may afflict individuals of any age, is characterized by the triad ofmicroangiopathic hemolytic anemia, thrombocytopenia and uremia.Currently in North America, HUS is the most frequent cause of acuterenal failure in infants and young children. In a study by Siegler etal. of 288 patients treated for postdiarrheal HUS in Utah from1970–1994, severe disease (defined as anuria lasting longer than 7 days,oliguria lasting for longer than 14 days, or extrarenal structuraldamage such as stroke) occurred in 25% of cases and was associated withchildren less than two years of age; about one third of these severecases of HUS resulted in death (5%) or severe sequelae includingend-stage renal disease (5%) or chronic brain damage (3–5%), with lesssevere chronic problems involving hypertension, proteinuria, orazotemia.

TTP, which most often affects adults, is characterized by neurologiccomplications such as stroke, in addition to thrombocytopenia, hemolyticanemia and renal disease.

By far the most common EHEC serotype is O157:H7. Nevertheless, otherEHEC serotypes also cause HUS and hemorrhagic colitis, includingO26:H11, O111:H8 and a number of others. EHEC strains associated withHUS always elaborate one or more Shiga toxins and carry a 60 MDavirulence plasmid. In addition, most also harbor a chromosomalpathogenicity island (so-called LEE) having a set of genes that encodethe ability to attach and efface. It is well accepted that Shiga toxinselaborated by EHEC play a key role in the pathogenesis of hemorrhagiccolitis and HUS.

As described in detail below, the Shiga toxin family is comprised of twogroups of toxins, Stx1(which is essentially identical tocytotoxin/neurotoxin/enterotoxin produced by Shigella dysenteriae type1, the Shiga bacillus) and Stx2 (which is immunologically distinct fromStx1 and has several related variants). In the USA, the overwhelmingmajority of EHEC associated with cases of HUS express Stx2, either aloneor in conjunction with Stx1.

The most important reservoir of EHEC infection are bovines. The singlemost important mode of transmission of EHEC to humans is via theconsumption of under-cooked contaminated beef, most often ground beef.Less commonly, a variety of other food vehicles and other modes oftransmission have been incriminated. Most notably, EHEC are one of thehandful of bacterial enteric pathogens, which, like Shigella, can betransmitted by direct contact or by contact with contaminated fomites.

There is great anticipation and optimism on the part of mostepidemiologists that irradiation of meat sold in the USA willdrastically curtail the transmission of EHEC to humans, since it willcurtail the single most important mode of transmission. Nevertheless,certain risk groups exposed to other modes of transmission of EHEC willnot benefit from this intervention. For example, the exposure ofabattoir workers to EHEC, an occupational hazard, occurs at a point inthe meat processing cycle prior to when irradiation would be utilized.For such special groups such as these for whom risk will remain evenafter irradiation of meat becomes commonplace, anti-EHEC vaccines can beuseful. The present invention provides vaccines against EHEC useful forthe prevention of infection (in the animal reservoirs or in humans) andfor preventing the severe complications of EHEC infection by stimulatingneutralizing Shiga antitoxin.

Studies with attenuated Vibrio cholerae O1 expressing Stx1 B subunithave demonstrated the feasibility of eliciting neutralizing Shigaantitoxin by mucosal immunization with live vectors. However, sincevirtually all EHEC associated with HUS cases in the USA express Stx2,alone or in conjunction with Stx1, it is preferable that a vaccine forpreventing the severe complications of EHEC infection via elicitation oftoxin-neutralizing antibodies should stimulate anti-Stx2 as well asStx1. It is within the broad scope of the present invention to provide astabilized plasmid system for expressing Stx2 antigens, alone or inconjunction with Stx1, in an attenuated S. typhi live vector.

Other antigens which may be suitably delivered according to thecompositions and methods of the present invention include, for example,those for hepatitis B, Haemophilus influenzae type b, hepatitis A,acellular pertussis (_(ac)P), varicella, rotavirus, Streptococcuspneumoniae (pneumococcal), and Neisseria meningitidis (meningococcal).See Ellis et al., Advances in Pharm., 39: 393423, 1997 (incorporatedherein by reference).

In one aspect, the antigens encoded by the expression plasmids of thepresent invention are cancer vaccines.

In another aspect, the antigens encoded by these plasmids are designedto provoke an immune response to autoantigens, B cell receptors and/or Tcell receptors which are implicated in autoimmune or immunologicaldiseases. For example, where inappropriate immune responses are raisedagainst body tissues or environmental antigens, the vaccines of thepresent invention may immunize against the autoantigens, B cellreceptors and/or T cell receptors to modulate the responses andameliorate the diseases. For example, such techniques can be efficaciousin treating myasthenia gravis, lupus erythematosis, rheumatoidarthritis, multiple sclerosis, allergies and asthma.

5.8.4.1 The Shiga Toxin Family

Conradi in 1903 first reported that S. dysenteriae 1 produced a powerfulexotoxin. Because injection of this toxin led to hind limb paralysis ofrabbits it was originally called a neurotoxin. Subsequently this toxin,Shiga toxin, was shown to be lethal for certain cells in tissue culture(i.e., it was a cytotoxin). Vicari et al. and then Keusch et al.demonstrated that it also functioned as an enterotoxin.

Scientists now recognize the existence of a family of Shiga cytotoxinswhich inhibit protein synthesis, leading to cell death for susceptiblecells. For many years after the revelation that such toxins wereproduced by certain E. coli strains in addition to the original Shigatoxin produced by Shigella dysenteriae type 1, the nomenclature for thisfamily of toxins was confusing. Since early reports described theactivity of these toxins on Vero cells (a cell line derived from Africangreen monkey kidney epithelial cells), many investigators called themverotoxins. Others referred to these toxins expressed in E. coli asShiga-like toxins.

The protein toxins are collectively referred to herein as Shiga toxins(Stx), and the genes encoding these toxins are designated as stx withsubscripts denoting the group and variant [i.e. stx₁ for the Shiga toxinproduced by E. coli that is essentially identical to that of Shigelladysenteriae type 1 (stx), and stx₂, Stx_(2c), Stx_(2d), Stx_(2e) for theantigenically distinct group of related toxins].

The structure, biochemistry and antigenicity of Shiga toxins are welldescribed in Melton-Celsa et al., Eschericia coli O157:H7 and OtherShiga Toxin-producing E. coli Strains, 1998; Takeda, Bacterial Toxinsand Virulence Factors in Disease, 1995; Gyles, Canadian J. ofMicrobiology, 38:734, 1992; and O'Brien et al., Current Topics inMicrobiology and Immunology, 180:165, 1992 (the disclosures of which areincorporated herein by reference).

These Shiga cytotoxins are composed of a single catalytic A subunit ofapproximately 32 kDa non-covalently associated with a pentamericreceptor binding domain of approximately 7.7 kDa B subunits. Thesesubunits are encoded by a single operon of the order stxA-stxB;transcription of the stx and stx₁ operons are iron-regulated in both S.dysenteriae type 1 and E. coli, but no environmental control signalshave as yet been determined for any stx₂ operon. None of these toxins isencoded on a plasmid; rather they are phage-encoded (Stx1, Stx2, Stx2c,and Stx2d) or are chromosomally encoded (Stx, Stx2e).

As mentioned above, all members of the Shiga toxin family are cytolytictoxins which inhibit protein synthesis within susceptible cells byblocking the binding of elongation factor 1-dependent aminoacyl-tRNA toribosomes. For all toxins identified from human infections, penetrationof susceptible cells by endocytosis follows binding of the holotoxin tothe necessary cell surface glycolipid receptor globotriaosyl ceramide(Gb₃), traffiking of the toxin to the Golgi apparatus and endoplasmicreticulum, followed by release into the cytoplasm. Shiga toxins are RNAN-glycosidases which depurinate a single adenine from the 28S RNA of theeukaryotic 60S ribosomal subunit, thus inactivating the 60S subunit andeventually leading to cell death.

There are six prototypic members of the Shiga toxin family: Stx, Stx1,Stx2, Stx2c, Stx2d, and Stx2e, which differ from one anotherimmunologically and in toxic activity. Significant detail has beenincluded here to provide background for understanding the significanceof point mutations discussed below, which are required for thegenetically detoxified holotoxins. The members of the Shiga toxin familydiffer from one another in 3 fundamental ways, as recently summarized byMelton-Celsa et al., Eschericia coli 0157:H7 and Other Shigatoxin-producing E. coli strains, 1998.

(1) Immunologically: The Shiga toxin family is composed of twoserogroups, Stx/Stx1 and Stx2; antisera raised against Stx/Stx1 do notneutralize members of the Stx2 serogroup, as judged by the Vero cellcytotoxicity assay.

(2) Structurally: Stx and Stx1 are essentially identical, differing in asingle amino acid at position 45 of the mature A subunit, and thecrystal structure for the Stx holotoxin has been solved. The prototypeStx2 is only 55% homologous to residues of the mature A subunit ofStx/Stx1 and 57% homologous to the mature B subunit, which explains whyantisera raised against Stx/Stx1 do not neutralize members of the Stx2group. Within the Stx2 group, Stx2e is most distantly related, sharing93% amino acid homology to the mature A subunit of Stx2 and 84% homologyto the mature B subunit; Stx2c and Stx2d are very similar to Stx2,sharing 99–100% homology in mature A subunit residues and 97% homologyin mature B subunit residues.

(3) Cytotoxicity: Stx2 is among the most lethal of the Shiga toxins,with an LD₅₀ for mice injected intraperitoneally of 0.5–2 ng. The LD₅₀for Stx1 and Stx2e is 200–400 ng, and 1–5 ng for Stx2d; however, Stx2dis unusual in that this toxin can become activated by murine intestinalmucus to increase the toxicity of the toxin, lowering the LD₅₀ to 0.5ng.

5.8.5 Site-Specific Mutagensis of Shiga Toxins

In one aspect, the invention provides a genetically detoxified Shigatoxin. The detoxification is accomplished by site-specific mutagenesis,introducing two defined and well-separated point mutations alteringcritical residues within the catalytic site of the A subunit. Theinvention also introduces two additional defined and well-separatedpoint mutations within the B subunit to alter critical residues withinthe primary binding site (i.e. SITE I) residing within the cleft formedby adjacent B subunits of the holotoxin pentameric ring.

Prior attempts have been made to alter the lower affinity binding SITEII. However, this binding site has only been identified from molecularmodeling studies, and is not extensively supported by mutational studieswhich favor SITE I binding of the Gb₃ receptor. Even if SITE II is analternate low-affinity binding site allowing entry of our mutantholotoxin into susceptible cells, the inactivation of the catalyticdomain will still prevent cell death.

Based on amino acid sequence alignments, X-ray crystallography studies,and molecular modeling studies, essential amino acids have beenidentified comprising the active site within the catalytic A subunit ofStx, as well as those residues comprising the binding SITE I within theB subunit pentamer of Stx/Stx1. It is the inventor's conclusion that theamino acids essential to the active site are selected from the groupconsisting of Tyr 77, Tyr 114, Glu 167, Arg 170, and Trp 203. Theresidues believed to be required for receptor binding to the cleftsformed by adjacent B subunits include Lys 13, Asp 16, Asp 17, Asp 18,Thr 21, Glu 28, Phe 30, Gly 60, and Glu 65. These site predictions areconsistent with functional studies and in vivo experiments using definedsingle and double mutations, within individual domains of the holotoxin,introduced by site-specific mutagenesis. A summary of such mutations ispresented in Table 1. Based on these data and crystallographicpredictions, it is within the broad practice of the invention to provideexpression plasmids encoding Shiga toxins having two specific sets ofpoint mutations within both the A and B subunits to create non-toxicmutant Stx2 holotoxins for use as vaccines, such as by expression withinattenuated S. typhi live vectors such as CVD908-htrA.

TABLE 1 SITE-SPECIFIC MUTAGENESIS STUDIES DROP IN DROP IN NEUTRALIZINGSUBUNIT TOXIN MUTATION CYTOTOXICITY LETHALITY ANTIBODIES A Stx1 Leu201 →Val + of NO cytotoxicity — — residues 202–213 Stx1 Glu167 → Asp 10³ — —Stx1 Arg170 → Leu 10³ — — Stx2 Glu167 → Asp 10³ — — Stx2e Glu167 → Asp10⁴ — — Stx2e Arg170 → Lys 10 — — Stx2e Glu167 → Asp 10⁴ — — Arg170 →Lys Stx2e Glu167 → Gln 10⁶ 10⁴ Y B Stx Asp16 → His + NO cytotoxicity — —Asp17 → His Stx Arg33 → Cys 10⁸ — — Stx Gly60 → Asp 10⁶ — — Stx1 Phe30 →Ala 10⁵ 10 Y Stx2 Ala42 → Thr 10³–10⁴ Y Y Stx2 Gly 59 → Asp 10³–10⁴ Y Y5.9 Pharmaceutical Formulations

It is contemplated that the bacterial live vector vaccines of thepresent invention will be administered in pharmaceutical formulationsfor use in vaccination of individuals, preferably humans. Suchpharmaceutical formulations may include pharmaceutically effectivecarriers, and optionally, may include other therapeutic ingredients,such as various adjuvants known in the art.

The carrier or carriers must be pharmaceutically acceptable in the sensethat they are compatible with the therapeutic ingredients and are notunduly deleterious to the recipient thereof. The therapeutic ingredientor ingredients are provided in an amount and frequency necessary toachieve the desired immunological effect.

The mode of administration and dosage forms will affect the therapeuticamounts of the compounds which are desirable and efficacious for thevaccination application. The bacterial live vector materials aredelivered in an amount capable of eliciting an immune reaction in whichit is effective to increase the patient's immune response to theexpressed mutant holotoxin or to other desired heterologous antigen(s).An immunizationally effective amount is an amount which confers anincreased ability to prevent, delay or reduce the severity of the onsetof a disease, as compared to such abilities in the absence of suchimmunization. It will be readily apparent to one of skill in the artthat this amount will vary based on factors such as the weight andhealth of the recipient, the type of protein or peptide being expressed,the type of infecting organism being combatted, and the mode ofadministration of the compositions.

The modes of administration may comprise the use of any suitable meansand/or methods for delivering the bacterial live vector vaccines to acorporeal locus of the host animal where the bacterial live vectorvaccines are immunostimulatively effective.

Delivery modes may include, without limitation, parenteraladministration methods, such as subcutaneous (SC) injection, intravenous(IV) injection, transdermal, intramuscular (IM), intradermal (ID), aswell as non-parenteral, e.g., oral, nasal, intravaginal, pulmonary,opthalmic and/or rectal administration.

The dose rate and suitable dosage forms for the bacterial live vectorvaccine compositions of the present invention may be readily determinedby those of ordinary skill in the art without undue experimentation, byuse of conventional antibody titer determination techniques andconventional bioefficacy/biocompatibility protocols. Among other things,the dose rate and suitable dosage forms depend on the particular antigenemployed, the desired therapeutic effect, and the desired time span ofbioactivity.

The bacterial live vector vaccines of the present invention may beusefully administered to the host animal with any other suitablepharmacologically or physiologically active agents, e.g., antigenicand/or other biologically active substances.

Formulations of the present invention can be presented, for example, asdiscrete units such as capsules, cachets, tablets or lozenges, eachcontaining a predetermined amount of the vector delivery structure; oras a suspension.

6. EXAMPLES

An isogenic series of expression plasmids composed of individualcassettes has been constructed for use in bacterial live vectorvaccines, such as E. coli and Salmonella. With the exception ofribosomal binding sites (RBS), the key genetic loci controllingtranscription initiation and termination, plasmid replication, orencoding expressed proteins are contained within defined restrictionfragments, as depicted by the representative plasmid diagram of pGEN2seen in FIG. 1A. The basic structure of these expression plasmids willfirst be highlighted and then the data demonstrating the function ofeach locus within the attenuated vaccine strain CVD908-htrA will besummarized.

6.1 pGEN Structure

Transcription of any heterologous antigen to be expressed withinCVD908-htrA is primarily controlled by an inducible promoter containedon an EcoRI-BglII cassette. Since the expression plasmids were initiallymodeled after pTETnir15, early versions carried theanaerobically-activated nir15 promoter (P_(nir15)). However, thispromoter has been replaced with a more tightly regulated osmoticallycontrolled promoter P_(ompC) which is easily manipulated in vitro byvarying the concentration of NaCl.

Heterologous antigens are contained on a BglII-AvrII cassette, flankedby an optimized RBS at the 5′-proximal end and a trpA transcriptionalterminator at the 3′-distal end of this cassette. The origin ofreplication for these expression plasmids has been designed as anAvrII-BglII cassette, and is protected from read-through transcriptionoriginating in flanking regions. These cassettes carry an extremelyefficient derivative of the T1T2 transcriptional terminator at oneterminus with the trpA transcriptional terminator from the heterologousantigen cassette at the opposite end of the replication cassette.

The flanking BglII and SpeI sites (see FIG. 2) between the replicationcassette and the selection cassette are intended for insertion of aplasmid maintenance function, such as the par locus from pSC101. Theselection cassettes contained within the plasmids are contained withinSpeI-XbaI cassettes, and can, for example, be used to encode resistanceto carbenicillin (the bla gene) or resistance to tetracycline (the tetAgene, see FIG. 1).

The drug resistance cassette can be replaced with the ssb gene encodingthe essential single stranded binding protein of Salmonella typhiCVD908-htrA.

The flanking XbaI and EcoRI sites between the selection cassette andP_(ompC) are intended for insertion of additional maintenance functions,including a PSK locus such as hok-sok (see FIGS. 1 and 2), or anadditional partition function such as the parA locus from pR1 (see FIG.7).

6.2 Modified ompC Promoter

It was intended that any promoter controlling transcription of aheterologous gene be responsive to an environmental signal of biologicalrelevance. For the expression plasmids described here, an ompC promotercassette (P_(ompC)) from E. coli was used, which is induced by increasesin osmolarity. Construction of this cassette was based on the publishedsequence of P_(ompC) published by Norioka et al (Norioka et al. 1986)and was carried out using synthetic primers to create a 459 bpEcoRI-BglII cassette in which the natural RBS was removed.

To confirm that this promoter was osmotically controlled within CVD908-htrA, a derivative of pTETnir15 was constructed in whichP_(nir15)-toxC was replaced by a cassette comprised of P_(ompC) drivingexpression of a promoterless aphA-2 cassette conferring resistance tokanamycin. This plasmid, designated pKompC, was introduced into CVD908-htrA by electroporation, and recipients were screened for resistanceto kanamycin on LB medium. The osmotically regulated expression ofaphA-2 was determined by inoculating CVD 908-htrA(pKompC) into 50 ml ofsupplemented nutrient broth (NB) containing increasing concentrations ofkanamycin from 0 to 300 μg/ml; a parallel set of cultures were set upwith the identical ranges of kanamycin added, but also containing 10%sucrose to induce P.sub.ompC_(ompC). Cultures were incubated overnightat 37° C., and the O.D.₆₀₀ was measured. Results are reported in theTable 2, Experiment 1.

TABLE 2 shows induction with osmolarity of the promoter P_(ompC),controlling expression of resistance to kanamycin, within the attenuatedS. typhi live vector CVD 908-htrA.

TABLE 2 EXPERIMENT 1¹ EXPERIMENT 2² Concen- Concen- tration of Low 10%tration of Low 300 mM kanamycin osmolarity sucrose kanamycin osmolarityNaCl (μg/ml) (O.D.₆₀₀) (O.D.₆₀₀) (μg/ml) (O.D.₆₀₀) (O.D.₆₀₀) 0 0.92 0.350 0.95 1.04 50 0.13 0.35 200 0.04 0.99 100 0.07 0.31 400 0.02 0.96 2000.03 0.21 600 0.01 0.92 300 0.02 0.19 800 0.01 0.92 ¹A culture ofCVD908-htrA(pKompC) was set up in LB broth supplemented with 0.0001%(w/v) 2,3-dihydroxybenzoic acid (DHB) and 50. μg/ml of kanamycin, andwas incubated for 16 hr at 37° C. This initial culture was then diluted1:10 into fresh medium and incubated at 37° C. for two hrs to provide aseed culture of exponentially growing bacteria. 50 μl of this culturewere then inoculated into 50 ml Nutrient Broth (NB)cultures supplementedwith DHB as above, but with increasing concentrations of kanamycin; aparallel set of cultures were set up with the identical ranges ofkanamycin added, but also containing 10% sucrose to hopefully induceP_(ompC). Cultures were incubated overnight at 37° C., and the O.D₆₀₀was measured. ²A culture of CVD908-htrA(pKompC) in supplemented LB brothand kanamycin was incubated for 16 hr at 37° C., diluted 1:10 into freshmedium, and incubated at 37° C. for two hrs to provide a seed culture ofexponentially growing bacteria. 100 μl aliquots of this culture werethen inoculated into 50 ml NB broth cultures containing increasingconcentrations of kanamycin from 200 to 800 μg/ml; a parallel setofcultures were set up containing 300 mM NaCl, and all cultures wereincubated at 37° C. for 16 hr. and the O.D.₆₀₀ was measured.

Regardless of selective pressure using kanamycin, the presence of 10%sucrose had an inhibitory effect on the growth of CVD 908-htrA(pKompC).However, the results suggested that E. coli P_(ompC) was osmoticallycontrolled when driving aphA-2 gene expression within CVD908-htrA(pKompC). To confirm this, CVD 908-htrA(pKompC) was inoculatedinto 50 ml of supplemented NB broth, containing increasingconcentrations of kanamycin from 200 to 800 μg/ml; a parallel set ofcultures was again set up containing 300 mM NaCl to induce P_(ompC).Cultures were incubated at 37° C. for 16 hr, and results are reported inTable 2, Experiment 2. It was confirmed that P_(ompC)-driven expressionof the aphA-2 gene within CVD 908-htrA confers resistance to kanamycinat levels up to 800 μg/ml in an osmotically regulated manner.

The aph gene cassette was then replaced with a 756 bp BglII-NheIcassette containing the gfpuv allele encoding GFPuv. During the visualscreening of E. coli colonies sub-illuminated with ultraviolet light,one very brightly fluorescing colony and another representativefluorescent colony were chosen for further study, designated clone 1 andclone 3, respectively. Upon purification of the plasmids involved, itwas determined that clone 1 contained a plasmid that no longer carried aBglII site separating P_(ompC) and gfpuv, while clone 3 carried theexpected BglII site. We examined the induction of GFP expression whenclones 1 and 3 are grown on nutrient agar in the presence or absence ofNaCl, and determined by visual inspection that clone 3 displayed verylittle fluorescence when grown on nutrient agar containing no NaCl butfluoresced brightly when plated on nutrient agar containing 300 mM NaCl(data not shown). Clone 1, however, had a higher background level offluorescence when uninduced, but fluoresced intensely when induced with300 mM NaCl. To rule out mutations within the gfpuv gene which mightaffect fluorescence, we replaced P_(ompC) from clone 1 with P_(ompC)from clone 3, and confirmed the expected decrease in fluorescence asjudged by sub-illumination (data not shown). We therefore concluded thatdifferences in observed fluorescence were controlled by two geneticallydistinct versions of the P_(ompC) promoter, which we designate asP_(ompC) (higher transcription levels with less osmotic control) andP_(ompC3) (moderate transcription levels with osmotic control similar tothat observed for the P_(ompC)-aph cassette described above); wedesignate the plasmids containing these expression cassettes aspGFPompC1 and pGFPompC3, respectively.

To quantify the differences in induced and uninduced expression of gfpuvcontrolled by P_(ompC1) and P_(ompC3) GFPuv synthesis was monitoredwithin both E. coli DH5α and S. typhi CVD 908-htrA using flow cytometry.This powerful technique has the unique advantages of allowing rapidmeasurement of GFPuv expression within large numbers of individualbacteria, as well as accurately determining the mean intensity offluorescence due to GFPuv synthesis within each bacterial populationanalyzed. To accomplish this, pGFPompC1 and pGFPompC3 were introduced byelectroporation, and colonies were isolated on supplemented 1×LB agarcontaining 100 μg/ml of carbenicillin grown at 30° C. for 48 hr.Isolated colonies were then grown up and cultures frozen down as masterstocks. Fresh colonies were then inoculated into either supplementednutrient broth or supplemented nutrient broth containing 150 mM NaCl,and grown at 37° C./250 rpm for 24 hr; the difference in O.D.₆₀₀ for anyculture was never greater than 0.07. Induction of expression of gfpuv,controlled by P_(ompC1) and P_(ompC3), was analyzed by flow cytometry,and results are presented in Table 3.

TABLE 3 shows a comparison of induction of P_(ompc1) and P_(ompC3),controlling expression of GFPuv, within the host strains E. coli DH5αand CVD 908-htrA.¹

TABLE 3 Mean Mean Low Fluores- 150 mM Fluores- Induc- osmolarity cenceNaCl cence tion STRAIN (O.D.₆₀₀) Intensity (O.D.₆₀₀) Intensity Ratio²DH5α 0.61 0.28 0.95 0.29 NA³ DH5α 0.56 4.45 0.72 7.69 1.7 (pGFPompC1)DH5α 0.58 1.77 0.73 4.21 2.4 (pGFPompC3) CVD 908-htrA 0.58 0.27 0.650.26 NA CVD 908-htrA 0.60 5.37 0.54 23.4 4.4 (pGFPompC1) CVD 908-htrA0.54 2.56 0.53 17.1 6.7 (pGFPompC3) ¹All strains were streaked fromfrozen master stocks onto 2X LB agar supplemented with DHB and 50 μg/mlof carbenicillin, and incubated for 36 hr at 30° C. Isolated colonieswere pooled into 300 μl of NB broth supplemented with DHB andcarbenicillin, from which 25 μl were inoculated into 25 ml supplementedNB broth, with and without 150 mM NaCl, and incubated at 37° C., 250 rpmfor 24 hr. Bacteria were then pelleted,resuspended in 1 ml PBS pH 7.4,and then diluted 1:1000 into PBS for analysis by flow cytometry.²Defined as the ratio of mean fluorescent intensity measured afterinduction with 150 mM NaCl, divided by basal level of mean fluorescentintensity measured at low osmolarity. ³NA = not applicable.

The basal level of expression for the P_(ompC1)-gfpuv cassette is 2.5times higher than for the P_(ompC3)-gfpuv cassette, when expressed inDH5α, and 2.1 times higher when expressed within CVD 908-htrA; however,the basal level of fluorescence detected for synthesis of GFPuv neverexceeded a mean fluorescent intensity of 5.37, regardless of hostbackground. If we define induction ratio as the ratio of meanfluorescent intensity measured after induction, divided by basal levelof mean fluorescent intensity, it was observed that when induced with150 mM NaCl, P_(ompC1) and P_(ompC3) displayed within DH5α inductionratios of 1.7 and 2.4 respectively. Surprisingly, the induction ratiofor P_(ompC1) when measured in CVD 908-htrA was 4.4, and produced amaximum mean fluorescence intensity of 23.4 for these experiments.Although the induction ratio for P_(ompC3) within CVD 908-htrA was 6.7,the mean fluorescence intensity of 17.1 was lower than measured forP_(ompC1). Based on these data, it appears that P_(ompC1) is thestrongest and yet osmotically controlled of the two ompC promoters.P_(ompC1) was therefore chosen for synthesis of the widest possiblerange of heterologous test antigen to examine the effects of suchsynthesis on plasmid stability.

These data clearly show that when driving expression of gfpuv within thelive vector strain CVD 908-htrA, P_(ompC1) and P_(ompC3) are induciblewith increasing osmolarity, although the basal level of transcription isstill noteworthy in both cases. The results observed under conditions oflow osmolarity further support our observations using solid media thatP_(ompC1) drives higher heterologous antigen expression than P_(ompC3).Since P_(ompC3) was noted to possess the intended 3′-terminal BglIIsite, which was not detected for P_(ompC1), we determined the nucleotidesequence for P_(ompC1) to perhaps detect point mutation(s) which mightexplain the strength of P_(ompC1). The only differences identified werelocated at the 3′-terminus of the cassette. The intended sequence withinthis region was 5′- . . . catataacAGATCTtaatcatccacAGGAGGatatctgATG-3′(SEQ ID NO: 4) (from left to right, upper case denotes the BglII site,ribosome binding site, and GFPuv start codon respectively); the actualsequence proved to be 5′- . . .catataacAGATCGATCTtaaAcatccacAGGAGGAtAtctgATG-3 (SEQ ID NO: 5) (insertedor changed bases denoted with underlined bold upper case). These changesdetected within the ompC1 promoter sequence are apparently responsiblefor increasing the observed strength of P_(ompC1) by an unknownmechanism, since neither the basic ompC promoter sequence, nor theoptimized ribosome binding site have been spontaneously altered.

6.3 Origins of Replication and Selection Cassettes

The success of expressing potentially toxic or otherwise problematicheterologous antigens within CVD908-htrA depends on the copy number ofthe expression plasmid. In addition, observed immune responses to agiven heterologous antigen are affected by the copy number of thegene(s) encoding the antigen, with chromosomally expressed antigenseliciting poorer immune responses when compared to plasmid-basedexpression.

An optimized immune response will depend on multicopy plasmid-basedexpression of the heterologous antigen(s) from plasmids with theappropriate copy number.

Since the appropriate copy number for a given heterologous gene cannotbe known a priori, the present invention provides a set of expressionplasmids which contain the origins of replication oriE1 (amplified frompAT153; copy number ˜60), ori15A (amplified from pACYC184; copy number˜15), and ori101 (amplified from pSC101; copy number ˜5). Theseself-contained replication cassettes are all carried on BglII-BamHIfragments, and are depicted for a set of 3 tetracycline-resistanceexpression plasmids shown in FIGS. 1A–1C.

Expression of the P_(ompC1)-controlled gfpuv expression cassettecontained on these expression plasmids was analyzed using flowcytometry. These experiments were designed to detect whether differencesin the level of observed fluorescence could be correlated with theexpected copy number of a given expression plasmid. CVD908-htrA strainscarrying pGEN2, pGEN3, and pGEN4 were streaked onto the rich mediumSuperAgar supplemented with DHB and 20 μg/ml tetracycline whereappropriate. SuperAgar was used because it is a very rich medium (3×LBagar). Plates were incubated at 30° C. to reduce the toxicity of GFPsynthesis and allow bacteria to grow luxuriously on the plates. Isolatedcolonies were then inoculated into 45 ml of SuperBroth supplemented withDHB and 20 μg/ml tetracycline where appropriate, and incubated at 37° C.for 16 hr. Bacteria were concentrated by centrifugation and resuspendedin 1 ml of sterile PBS, pH=7.4, and diluted 1:100 in PBS, pH=7.4 priorto FACS analysis. Bacteria were analyzed by flow cytometry, as describedabove, for two independent growth experiments, and results are displayedin Table 4 at the end of this section.

These data support the conclusion that overexpression of GFPuv withinCVD908-htrA is toxic to the bacteria. As the theoretical copy numberincreases for the plasmids pGEN4, pGEN3, and pGEN2 expressing GFPuvunder identical growth conditions from the identical P_(ompC1) promoter,the percentage of the growing population which fluoresces declines. Itis expected that the “dim” bacteria are not viable bacteria and may nolonger contain the expression plasmid, since these cultures were grownin the presence of 20 μg/ml tetracycline. It is noted, however, thatwhen streaked onto solid medium and grown at 37° C. for 24–36 hr,CVD908-htrA(pGEN2) grows poorly and fails to produce isolated colonies,while CVD908-htrA(pGEN3) and CVD908-htrA(pGEN4) grow quite well andproduce intensely fluorescing isolated colonies.

GFPuv is employed herein as representative of other problematicheterologous antigens which would be of interest to include in abacterial live vector, such as the S. typhi-based live vector; however,it will be appreciated that GFPuv can be replaced by any non-metabolicprotein or peptide antigen.

The data above show that although use of medium-copy expression plasmidscontaining oriE1 replicons can be of use in expression of some antigens,expression of antigens of higher toxicity will be more successfullyexpressed from lower copy number plasmids which employ origins ofreplication yielding average copy numbers between 2 and 30, such asori101 or ori15A origins of replication.

TABLE 4 Experiment 1 Experiment 2 Mean Mean Fluorescence Fluorescence OfDim Of Dim Percent Bacteria Percentb Mean Percent Bacteria Percent MeanDim (Relative Fluorescing Fluorescence Dim (Relative FluorescingFluorescence Strain Bacteria Units) Bacteria (Relative Units) BacteriaUnits) Bacteria (Relative Units) CVD908-htrA 100 0.6 0 0 100 0.3 0 0CVD908- 19.9 0.1 80.1 38.5 37.2 0.3 62.8 10.1 htrA(pGEN2) CVD908- 17.10.1 82.9 28.1 4.9 0.2 95.1 8.28 htrA(pGEN3) CVD908- 12.1 0.1 88.0 22.49.4 0.3 90.6 4.25 htrA(pGEN4)6.4 The hok-sok Antisense Post-Segregational Killing Locus

Using the polymerase chain reaction, the hok-sok PSK genes wereamplified using the multiple antibiotic resistance R-plasmid pR1 as thetemplate in these reactions. All initial attempts to clone this locusonto either high or medium copy number plasmids were unsuccessful. Inorder to directly select for the hok-sok locus during subcloning, a setof primers was designed for use in overlapping PCR reactions such thatthe final product was a fragment containing a genetic fusion of thehok-sok locus from pR1 and a promoterless tetA gene from pBR322 encodingresistance to tetracycline. This cassette was engineered such thattranscription of the hok gene would continue into tetA; the two lociwithin this cassette were separated by an XbaI restriction site forfuture manipulations.

Construction of this cassette not only allowed for direct selection ofthe hok-sok locus, but also allowed for confirmation that the PSKfunction would operate in S. typhi CVD908-htrA. After electroporation ofplasmids carrying the cassette into CVD908-htrA, transformants could beselected using tetracycline. Successful recovery of isolated coloniesindicates successful synthesis of the hok-tetA mRNA, and successfulsynthesis of the antisense sok RNA to prevent translation and synthesisof Hok, which would kill the bacteria. Recovery of the hok-sok-tetAcassette then became straightforward, and was easily incorporated intoour expression plasmids to create the selectable marker cassette of theplasmids pGEN2, pGEN3, and pGEN4 depicted in FIGS. 1A–1C.

Experiments were then initiated to determine the effect of the hok-sokPSK function on the stability of expression plasmids containing oriE1and the resistance marker bla encoding β.-lactamase which confersresistance to carbenicillin. The hok-sok cassette was inserted into thepAT153-based expression plasmid pTETnir15, in which the Pnir15-toxCheterologous antigen cassette was replaced with our P_(ompC1)-gfpuvcassette, creating the plasmids pJN72 (without hok-sok) and pJN51 (withhok-sok). An additional set of plasmids was created by replacingP.sub.ompC1 with the weaker promoter P_(ompC3), creating pJN10 andpJN12; the structures of these four isogenic plasmids are represented inFIG. 2. CVD908-htrA strains carrying either pJN72, pJN5 1, pJN10, orpJN12 were streaked onto the rich medium SuperAgar supplemented with DHBand 100 μg/ml carbenicillin, and plates were incubated as above for thepGEN plasmids at 30° C. to reduce the toxicity of GFPuv synthesis andallow bacteria to grow luxuriously on the plates.

Isolated colonies were then inoculated into 45 ml of Super brothsupplemented with DHB and 100 μg/ml carbenicillin and grown at 37° C.for 24 hours for analysis by flow cytometry of fluorescence. A secondindependent experiment was carried out exactly as the first, exceptisolated colonies were suspended in 500 μl of Super broth and 250 μleach inoculated into 45 ml paired Super broth cultures with or without300 mM NaCl added to induce the P_(ompC)-gfpuv cassettes; cultures wereincubated at 37° C. for 48 hrs and again analyzed by flow cytometry; andresults for both experiments are displayed in Table 5. Fluorescencehistograms for uninduced and induced expression plasmids from experiment2 are represented in FIGS. 3A–3H.

TABLE 5 Experiment 1 Experiment 2 Mean Mean Percent Fluorescence PercentFluorescence Dim Of Dim Fluorescing Mean +/−300 mM % Dim Of Dim %Fluorescing Mean Strain Bacteria Bacteria Bacteria Fluorescence OD.₆₀₀NaCl Bacteria Bacteria Bacteria Fluorescence CVD908-htrA 100 0.3 0.73 −100 0.3 0 0 CVD908- 3.1 0.2 96.9 10.2 0.75 − 2.3 0.3 97.7 11.7htrA(pJN72) 0.89 + 22.2 0.3 77.8 22.5 CVD908- 58.1 0.3 41.9 6.29 0.62 −56.3 0.3 43.7 18.4 htrA(pJN72) 0.82 + 95.4 0.3 4.6 21.0 CVD908- 5.4 0.294.6 7.43 0.72 − 1.7 0.3 98.3 8.3 htrA(pJN10) 0.96 + 29.9 0.3 70.1 19.8CVD908- 18.9 0.2 81.1 6.60 0.47 − 45.2 0.3 54.8 16.4 htrA(pJN12) 0.68 +95.6 0.3 4.4 13.2

These flow cytometry results can be explained as follows: expression ofGFPuv (or other potentially detrimental heterologous antigen) from amulticopy expression plasmid such as pJN72 increases the metabolicstress on the CVD 908-htrA(pJN72) live vector, and increases plasmidinstability in the absence of selection. Since the selectable marker ofthe expression plasmid encodes the secreted enzyme .beta.-lactamase,then as time increases the concentration of carbenicillin in thesurrounding medium declines, selective pressure decreases, and thefrequency of plasmid loss increases; however, since multicopy plasmidsare involved, relatively few bacteria succeed in losing all residentplasmids, but the average copy number of pJN72 per bacterium drops.

Quantitation by flow cytometry of GFPuv production for an uninducedpopulation of healthy growing CVD 908-htrA(pJN72) indicates that themajority of bacteria express GFPuv and few non-fluorescing cells aredetected (FIG. 3A). However, increasing production of GFPuv by inductionof the P_(ompC1)-gfpuv cassette increases the metabolic stress on CVD908-htrA(pJN72), and although the production of GFP doubles, thepercentage of non-fluorescent bacteria increases as more plasmids arelost from the population (FIG. 3B).

In a similar population of growing CVD 908-htrA(pJN51), each bacteriumcarries multicopy plasmids encoding both GFPuv and a PSK function. Thefrequency of plasmid loss for pJN51 remains the same as for pJN72, butin this case as individual bacteria lose copies of the expressionplasmid, the 1:1 stoichiometry between the mRNA levels of hok and sok isdisturbed, and production of Hok leads to cell death; therefore, theonly CVD 908-htrA(pJN51) bacteria that will grow rapidly will be thosewhich retain all of their expression plasmids. Accordingly, it is notsurprising that quantitation by flow cytometry of GFPuv production foran uninduced population of healthy growing CVD 908-htrA(pJN51) nowdetects a population of fluorescing bacteria which displays levels ofGFPuv fluorescence equivalent to those observed for CVD 908-htrA(pJN72)grown under inducing conditions (FIG. 3C vs FIG. 3B); however, thepercentage of non-fluorescing bacteria rises to over half the overallpopulation of organisms.

Increasing production of GFPuv in this population by induction of theP_(ompC1)-gfpuv cassette in CVD 908-htrA(pJN5 1) again increases themetabolic stress on the live vector, but now the percentage ofnon-fluorescent bacteria almost completely overtakes the few fluorescingbacteria as many plasmids are presumably lost from the population andbacteria are killed (FIG. 2D).

One would expect that if a weaker promoter is used to control expressionof GFPuv, the overall fluorescence of the population would be decreased(compared to that observed for a similar population of organisms grownwith a strong promoter expressing GFPuv under identical conditions), andthe percentage of non-fluorescent bacteria should drop due to theoverall drop in GFPuv synthesis. However, as seen in FIGS. 3E–3H, use ofthe weaker P_(ompC3)-gfpuv cassette did not significantly improve theviability of induced bacteria carrying a killing system, even thoughoverall expression of GFPuv was reduced.

It is concluded that in order to maximize the percentage of a populationof live vectors expressing the heterologous antigen of choice, it is notsufficient only to incorporate a PSK function into a given expressionplasmid, whether it be a drug resistance marker, the asd system, analternate ssb system, or the hok-sok killing system. In addition tooptimizing copy number and expression levels, the segregationfrequencies of these plasmids must also be improved to ensure that eachdaughter cell in an actively growing population will inherit at leastone expression plasmid and those that do not will be killed and removedfrom the population. It is therefore within the scope of the presentinvention to provide an expression plasmid having a PSK function andfurther having optimized copy number and/or expression levels, coupledwith incorporation of one or more SEG functions.

6.5 Complementation-Based Killing System

It is also within the broad scope of the present invention to provide anexpression plasmid comprising a complementation-based killing system,for example, a system involving the deletion of the chromosomal ssblocus of CVD908-htrA by homologous recombination, andtrans-complementation of this lesion using multicopy plasmids carryingfunctional ssb.

To carry out such constructions requires cloning the relevant section ofthe S. typhi chromosome encompassing the ssb gene and flankingsequences, into which specific deletions can be introduced forchromosomal mutagensis.

Since our original submission, substantial progress has been made in thesequenceing of the Salmonella typhi chromosome at the Sanger Centre inLondon. The Sanger Centre is a genome research center set up in 1992 bythe Wellcome Trust and the Medical Research Council in order to furtherour knowledge of genomes. Among other projects, the Sanger Centre issequencing the 4.5 Mb genome of S. typhi, in collaboration with GordonDougan of the Department of Biochemistry, Imperial College, London. Theyare sequencing strain CT18, a highly pathogenic, multiple drug resistantstrain isolated from a typhoid patient in Cho Quan Hospital, Ho Chi MinhCity, Vietnam. This strain is known to harbor pVN100 (a 130 kb multidrugresistance plasmid) and a cryptic 80 kb plasmid. The genome is beingsequenced by a whole genome shot gun approach using a 2 kb pUC library,generated inhouse from chromosomal DNA supplied by Prof. Dougan's lab.Each insert is being sequenced once from each end. The shotgun phase isnow complete, and finishing has begun. At present there are 60 contigsover 1 kb in the database; a total of 5.106 Mb of sequence assembledfrom 87,331 reads.

Based on updated results posted Oct. 4, 1999, we have identified Contig343, which contains the S. typhi ssb locus and critical flankingsequences within a 205,199 bp region. We have designed primers 1 and 4(listed below) to amplify by PCR a 3535 bp fragment of the S. typhichromosome in which the ssb locus is flanked by 1.5 kb of chromosomalsequence; this flanking symmetry is required for optimal crossoverfrequenceis to introduce the counter-selectable sacB-neo cassette andreplace ssb. Using the methodology previously filed, we will use primers1 and 2 to engineer a 5′-proximal 1.5 kb Eco RI-Xma I cassette, upstreamof ssb. Primers 3 and 4 will be used to generate the 3′-distal 1.5 kbXma I-Eco RI cassette, downstream of ssb; both 1.5 kb cassettes will beligated together, forming the 3 kb Eco RI fragment containing a uniqueXma I site exactly in the middle of the cassette. The sacB-neo cassettecan now easily be inserted into the Xma I site, to complete constructionof the mutagenesis cassette to be inserted into pCON (previouslydescribed in our first filing). The required complementing ssb-1cassette will be constructed using primers 5 and 6 as a Nhe I cassettefor replacement of drug resistance markers within the Xba I-Spe Icassettes of pGEN 211, pGEN 222, pGEN 206, or any later version of theexpression plasmids detailed herein.

(SEQ ID NO: 6) PRIMER 1:5′-gaattcGCGCGCTTCGCGATTCAGTCGCGTTCCTTCACAGCTGGCGCAGGGGCGATTACTGATGAA-3′ (SEQ ID NO: 7) PRIMER 2:5′-cccggGAGTCTCCTGAATACGTTTCATAAAAGTGTAAACGCGTGAGT GTACCATTTCCACGTAGC-3′(SEQ ID NO: 8) PRIMER 3:5′-cccggGTAAAAAACTCAAAGCGTTATTTGCATTTTCGCTATAGTTCTCGTCTGCTGAAATGCCTGGTGT-3′ (SEQ ID NO: 9) PRIMER 4:5′-gaattcCATTTCTATCAATAAATTACTATTAGTTTTGTCTTCTAACCAAGCCTCTATTTTATGAGTATCCTCTTCAG-3′ (SEQ ID NO: 10) PRIMER 5:5′-gctagcATGGCCAGCAGAGGCGTAAACAAGGTGATTCTCGTTGGTAATCTGGGCCAGGACCCGGAAGTACGC-3′ (SEQ ID NO: 11) PRIMER 6:5′-gctagcTCAGAACGGAATGTCGTCGTCAAAATCCATTGGCGGTTCGT TAGACGGCGCTGGCGCG-3′6.6 Stability of Expression Plasmids in the Absence of Selection

In order to develop a non-catalytic plasmid maintenance system toenhance the stability of multicopy expression plasmids encoding foreignantigens within CVD 908-htrA, experiments were initiated to monitorplasmid stability by quantitating expression of GFPuv by flow cytometrywhen strains were passaged in the absence of antibiotic selection. Theseexperiments were designed to address 3 fundamental questions: 1] What isthe effect of the induction level of P_(ompC1) on the stability ofplasmids encoding a heterologous antigen such as GFPuv? 2] What is theeffect of copy number on the stability of plasmids expressing GFPuv? 3]How do the hok-sok, par, and parA maintenance functions affect plasmidretention, both as individual components and synergistically?

Initial flow cytometry experiments were carried out in which CVD908-htrA carried replicons with either the oriE1, ori15A, or ori101origin of replication. It was quickly determined that replicons carryingthe higher copy number oriE1 origins were very unstable, even whenstrains were grown in the presence of antibiotic selection. Flowcytometry results indicated that even when cultured in the presence ofcarbenicillin, the percentage of the bacterial populations no longerexpressing detectable GFPuv ranged from approximately 50% for pGEN71(carrying hok-sok) and pGEN84 (hok-sok+par) to 62% for pGEN211(hok-sok+par+parA). Since replicons carrying an oriE1 origin clearly didnot allow for optimal synthesis of the heterologous GFPuv test antigenwithin the majority of a growing population of live vector bacteria,this series of expression plasmids was not examined further.

CVD 908-htrA carrying expression plasmids with an ori15A origin werethen examined. Strains were inoculated into 25 ml cultures of 1×LB+DHB(no antibiotic selection) containing either 50 mM, 150 mM, or 300 mMNaCl. Cultures were incubated for 24 hr at 37° C./250 rpm, diluted1:1000 into fresh medium of identical osmolarity, and incubated foranother 24 hr; samples from all cultures were analyzed for levels ofGFPuv synthesis by flow cytometry. Results for the first passage in theabsence of selection are listed in Table 6, and the histogramsrepresenting these data are shown in FIG. 8.

TABLE 6 shows stability within CVD 908-htrA of ori15A replicons,containing plasmid maintenance systems of increasing complexity, grownwithout selection and in the presence of increasing osmolarity.¹

TABLE 6 50 mM NaCl 150 mM NaCl 300 mM NaCl Percent Mean Percent MeanPercent Mean Fluorescing Fluorescence Fluorescing FluorescenceFluorescing Fluorescence STRAIN² O.D.₆₀₀ Bacteria Intensity O.D.₆₀₀Bacteria Intensity O.D.₆₀₀ Bacteria Intensity CVD908-htrA 0.98 100 0.61.11 100 0.6 1.12 100 0.6 pGEN91 1.00 13.2 28.6 1.17 11.4 42.9 1.26 10.965.5 pGEN111 1.26 47.4 51.8 1.17 28.9 93.6 1.12 42.4 65.1 pGEN121 1.0180.5 53.3 1.20 73.8 74.0 1.15 56.7 105.3 pGEN193 1.11 71.4 50.9 1.2465.2 64.7 1.22 53.7 90.8 pGEN222 1.01 96.8 52.1 1.28 93.3 67.8 1.13 95.389.2 ¹These data are represented as histograms in FIG. 8. ²All strainswere streaked from frozen master stocks onto 2X LB agar supplementedwith DHB and 50 μg/ml of carbenicillin, and incubated for 36 hr at 30°C. Isolated colonies were pooled into 300 μl of 1X LB broth supplementedwith DHB, from which 25 μl were inoculated into 25 ml of 1X LB brothcontaining DHB and either 50 mM, 150 mM, or 300 mM NaCl; cultures wereincubated at 37° C.,250 rpm for 24 hr. For the results presented in thistable, bacteria were then pelleted, resuspended in 1 ml PBS pH 7.4, andthen diluted 1:1000 into PBS for analysis by flow cytometry.

In general, as osmolarity increases and induction of P_(ompC1) rises,the percentage of the live vector population expressing GFPuv drops;nevertheless, the mean level of fluorescence intensity increases asexpected. For example, in the presence of 50 mM NaCl, 80.5% of apopulation of CVD 908-htrA(pGEN121) express GFPuv with a meanfluorescence intensity of 53.3. As the concentration of NaCl increasesto 300 mM NaCl, the percentage of the population expressing GFPuv dropsto 56.7%; nevertheless, the mean fluorescence intensity rises to 105.3.However, it is notable that for strains carrying pGEN222 with a completeplasmid maintenance system (i.e hok-sok+par+parA), the percentage of thepopulation expressing the heterologous antigen remains at approximately95%, while the mean fluorescence intensity increases from 52.1 (50 mMNaCl) to 89.2 (300 mM NaCI). It was noted that upon further passage ofthese strains for an additional 24 hrs in the absence of antibioticselection, less than 5% of bacteria continued to express functionalGFPuv. Streaks of these cultures onto solid medium, prior to flowanalysis, indicated that non-fluorescing bacteria remained viable, butwere sensitive to antibiotic selection. When non-fluorescing bacteriawere sorted and plated, they were confirmed to be sensitive toantibiotic and non-fluorescent when irradiated with ultraviolet light,indicating loss of resident plasmids.

A passage experiment involving CVD 908-htrA carrying expression plasmidswith an ori101 origin detected no significant loss of GFPuv expressionafter passage of strains for 48 hrs without selection, regardless ofosmolarity. Therefore, strains were passaged in a separate experimentfor 96 hrs (i.e. 4×24 hr) in the presence of either 50, 150, or 300 mMNaCl. Populations were analyzed by flow cytometry after 3 and 4passages, and results are recorded in Table 7.

TABLE 7 shows stability within CVD 908-htrA of ori101 replicons,containing plasmid maintenance systems of increasing complexity, grownwithout selection and in the presence of increasing osmolarity.

TABLE 7 50 mM NaCl 150 mM NaCl 300 mM NaCl Percent Mean Percent MeanPercent Mean STRAIN (Passage Fluorescing Fluorescence FluorescingFluorescence Fluorescing Fluorescence Number)¹ O.D.₆₀₀ BacteriaIntensity O.D.₆₀₀ Bacteria Intensity O.D.₆₀₀ Bacteria IntensityCVD908-htrA (#3) ND² 100 0.6 ND 100 0.5 ND 100 0.5 CVD908-htrA (#4) 1.00100 0.3 1.18 100 0.3 1.19 100 0.3 pGEN132 (#3) ND 45.5 29.0 ND 33.2 36.9ND 81.3 47.3 pGEN132 (#4) 1.03 10.9 27.8 1.20 7.6 36.1 1.32 51.3 47.5pGEN142 (#3) 1.05 99.5 35.5 1.23 98.9 45.1 1.28 96.5 47.8 pGEN142 (#4)1.17 94.4 38.0 1.29 91.5 45.0 1.33 93.9 47.7 pGEN206 (#3) 1.08 98.1 36.21.25 94.5 42.8 1.29 95.2 47.4 pGEN206 (#4) 1.13 80.2 32.6 1.26 68.6 36.61.33 93.5 41.3 ¹All strains were streaked from frozen master stocks onto2X LB agar supplemented with DHB and 50 μg/ml of carbenicillin, andincubated for 36 hr at 30° C. Isolated colonies were pooled into 300 μlof 1X LB broth supplemented with DHB, from which 25 μl were inoculatedinto 25 ml of 1X LB broth containing DHB and either 50 mM, 150 mM, or300 mM NaCl; cultures were incubated at 37° C., 250 rpm for 24hr(defined here as passage #1). For passage #2, 25 μl from passage #1were inoculated into 25 ml (i.e. 1:1000 dilution) of identical mediumand incubated at 37° C., 250 rpm for an additional 24 hr withoutselection. Passages 3 and 4 were carried out in identical fashion, butafter the next passage had been set up the remaining #bacteria were thenpelleted, resuspended in 1 ml PBS pH 7.4, and thendiluted 1:1000 intoPBS for analysis by flow cytometry. ²ND = not done.

Live vectors carrying unstabilized ori101 replicons eventually lost thecapacity to synthesize the heterologous antigen after 96 hr. Forexample, after 96 hr growth in the presence of 50 mM NaCl, only 10.9% ofCVD 908-htrA(pGEN132) expressed GFPuv and fluoresced. As theconcentration of NaCl in the medium was increased to 150 mM,fluorescence was detected in only 7.6% of the population; curiously, at300 mM NaCl, the percentage recovered to 51.3% fluorescing bacteria.Remarkably, CVD 908-htrA carrying either pGEN142 (hok-sok) or pGEN206(hok-sok+parA) retained synthesis of GFPuv in greater than 95% of thepopulation after 3 passages (72 hr), regardless of osmolarity (see Table7). The percentage of fluorescing CVD 908-htrA (pGEN142) remained nearthis level after 4 passages (96 hr), while decreasing slightly for CVD908-htrA (pGEN206).

Taken together, these data show that as copy number is reduced, theapparent stability of resident plasmids and proficiency of a live vectorto synthesize a heterologous antigen such as GFPuv increases; as plasmidmaintenance systems accumulate within a given plasmid, apparentstability and antigen synthesis are further enhanced. In addition, asthe induction of P_(ompC1) and concomitant production of theheterologous antigen increases, the percentage of a growing populationremaining capable of synthesizing antigen can be dramatically reduced.

6.7 Bacterial Strains and Culture Conditions

All plasmid constructions were recovered in Escherichia coli strain DH5αor DH5αF′IQ (Gibco BRL). Construction of the hok-sok gene cassette usedpR1 template DNA isolated from E. coli strain J53(pR1), a generous giftfrom James B. Kaper. The live vector S. typhi CVD 908-htrA is anauxotrophic derivative of the wild type strain Ty2 with deletions inaroC, aroD, and htrA (Tacket et al. 1997b). All strains used forexamination of plasmid stability were grown in media supplemented with2,3-dihydroxybenzoic acid (DHB) as previously described (Hone et al.1991; Galen et al. 1997). When grown on solid medium, plasmid-bearingstrains of CVD 908-htrA were streaked from frozen (−70° C.) masterstocks onto 2×Luria-Bertani agar containing (per liter) 20 g Bactotryptone, 10 g Bacto yeast extract, and 3 g NaCl (2×LB agar) pluscarbenicillin at a concentration of 50 μg/ml. Plates were incubated at30° C. for 24–36 hr to obtain isolated colonies ˜2 mm in diameter;strains were incubated at 30° C. to minimize the toxicity of GFPuvexpression in CVD 908-htrA.

When grown in liquid medium, cultures were incubated at 37° C., 250 rpmfor 16–24 hr. To examine the osmotic induction of the ompC promoter(P._(ompC)) within either E. coli DH5α or CVD 908-htrA, strains weregrown in Bacto nutrient broth (Difco) containing DHB and either NaCl orsucrose; cultures were supplemented either with 50 μg/ml ofcarbenicillin or increasing concentrations of kanamycin whereP_(ompC)-aphA-2 cassettes were examined. For quantitation of GFPuvsynthesis using flow cytometry, 6–8 isolated colonies from master stocksstreaked onto 2×LB agar as above were inoculated into 25 ml of 1×LBbroth supplemented with 50 μg/ml carbenicillin where desired and NaCl atincreasing concentrations to increase the induction of ompC promoters.Cultures were incubated at 37° C., 250 rpm for 16–24 hr prior topelleting bacteria for flow cytometry as described below.

6.8 Molecular Genetic Techniques.

Standard techniques were used for the construction of the plasmidsrepresented here (Sambrook et al., 1989). Unless otherwise noted, nativeTaq DNA polymerase (Gibco BRL) was used in polymerase chain reactions(PCR). S. typhi was prepared for electroporation of recombinant plasmidsafter harvesting from Miller's LB broth (Gibco BRL) supplemented withDHB; after pelleting bacteria, the cells were washed thrice with oneculture volume of sterile distilled water and resuspended in steriledistilled water to a final volume of 1/100 of the original culturevolume. Electroporation of strains was performed in a Gene Pulserapparatus (Bio-Rad) set at 2.5 kV, 200 Ω, and 25 μF. Followingelectroporation, bacteria were repaired using SOC medium and incubatingat 37° C., 250 rpm for 45 min; bacteria were then plated on 1×LB mediumcontaining DHB plus 50 μg/ml carbenicillin, and incubated at 30° C. for24 hr. Isolated colonies were then swabbed onto supplemented 2×LB andincubated at 30° C. for 16 hr. Frozen master stocks were prepared byharvesting bacteria into SOC medium without further supplementation andfreezing at −70° C.

6.9 Construction of Expression Vectors

The expression vectors listed in the following Table 8 were prepared inthe course of the recent work.

TABLE 8 Size Plasmid (kb) Relevant genotype Reference pTETnir15 3.7oriE1 toxC bla Oxer et al. (1991) pJN1 1.9 oriE1 bla This work pJN2 3.4oriE1 toxC bla This work pGFPuv 3.3 pUC19ori gfpuv bla Clontech pGFPompC3.5 oriE1 gfpuv bla This work pNRB1 3.5 oriE1 gfpuv tetA This work pGEN24.2 oriE1 gfpuv tetA hok-sok This work pGEN3 4.1 ori15A gfpuv tetAhok-sok This work pGEN4 5.6 ori101 gfpuv tetA hok-sok This work pJN5 3.1oriE1 gfpuv bla This work pJN6 3.7 oriE1 gfpuv bla hok-sok This workpJN7 4.1 oriE1 gfpuv bla hok-sok par This work pJN8 5.4 oriE1 gfpuv blahok-sok parA This work pGEN51 3.6 oriE1 gfpuv bla This work pGEN71 4.2oriE1 gfpuv bla hok-sok This work pGEN84 4.5 oriE1 gfpuv bla hok-sok parThis work pGEN183 5.9 oriE1 gfpuv bla hok-sok parA This work pGEN211 6.2oriE1 gfpuv bla hok-sok par parA This work pGEN91 3.5 ori15A gfpuv blaThis work pGEN111 4.1 ori15A gfpuv bla hok-sok This work pGEN121 4.5ori15A gfpuv bla hok-sok par This work pGEN193 5.8 ori15A gfpuv blahok-sok parA This work pGEN222 6.2 ori15A gfpuv bla hok-sok par parAThis work pGEN132 4.8 ori101 gfpuv bla par This work pGEN142 5.4 ori101gfpuv bla par hok-sok This work pGEN206 7.1 ori101 gfpuv blapar hok-sokpar A This work6.9.1 Construction of pJN1 and pJN2

The expression plasmids constructed for these studies are composed of 3basic cassettes encoding 1] expression of a heterologous antigen, 2] aplasmid origin of replication, and 3] selection and maintenancefunctions. To accomplish this, a basic replicon was constructed in whichthese cassettes were separated by unique restriction sites. The primersused in construction of the plasmid cassettes are set forth in thefollowing Table 9:

TABLE 9 GenBank Region of Primer Cassette Accession Region of Complemen-number Sequence¹ created Number Homology² tarity³ 15′-GCAGGAAAGAACATGTGA (SEQ ID NO: 12) oriE1 J01749 2463–2507GCCTAGGGCCAGCAAAAGGCC AGGAAC-3′ 2 5′-CATGACCAAAATCCCTTA (SEQ ID NO: 13)″ ″ 3197-3145 ACTAGTGTTTTAGATCTACTG AGCGTCAGAC CCCG-3′ 35′-CGGGGTCTGACGCTCAGT (SEQ ID NO: 14) bla ″ 3145–3197AGATCTAAAACACTAGTTAAG GGATTTTGGTCATG-3′ 4 5′-CTGTCAAACATGAGAATT (SEQ IDNO: 15) ″ ″ 17-1, CTAGAAGACGAAAGGGCCTCG 4361-4330 TGATACGCC-3′ 55′-ACAGCCTGCAGACAGATC (SEQ ID NO: 16) aphA-2 V00618 1–64CTTGACAGCTGGATCGCACTC TGGTATAATTGGGAAGCCCTG CAAAG -3′ 65′-GAAGCCCAACCTTTCATA (SEQ ID NO: 17) ″ ″ 1044-986 GAAGCTAGCGGTGGATCCGAAATCTCGTGATGGCAGGTTG-3′ 7 5′-AACAAGCGTTATAGGAAT (SEQ ID NO: 18) PompCK00541 4–33 TCTGTGGTAGCA-3′ 8 5′-ACTTTCATGTTATTAAAG (SEQ ID NO: 19) ″ ″498-469 ATCTGTTATATG-3′ 9 5′-AGATCTTAATCATCCACA (SEQ ID NO: 20) gfpuvU62636 289–317 GGAGGCTTTCTGATGAGTAAA GGAGAAGAACTTTTCACTG G-3′ 105′-GCTAGCTCATTATTTGTA (SEQ ID NO: 21) ″ ″ 1008-983 GAGCTCATCCATGG-3′ 115′-AGATGTGAATTCTAGATC (SEQ ID NO: 22) tetA J01749 4–41ATGTTTGAGAGCTTATCATCG ATAAGCTLTAATGCG-3′ 12 5′-AGATCTTATCAGGTCGAG (SEQID NO: 23) ″ ″ 1275-1234 GTGGCCCGGCTCCATGCACCG CGACGCAACGCG-3′ 135′-CGCGAATTGTCGAGACAA (SEQ ID NO: 24) hok-sok- X05813 2–48ACTCCGGGAGGCAGCGTGATG tetA CGGCAACAATCACACGGATTT C-3′ 145′-ATGAGCGCATTGTTAGAT (SEQ ID NO: 25) ″ J01749, 108-86,TTCATTTTTTTTTCCTCCTTA X05813 580-559 TTTTCTAGACAACATCAGGAA GGAGAAAGG-3′15 5′-CCTTTCTCCTTGCTGATG (SEQ ID NO: 26) ″ X05813, 559–580,TTGTCTAGAAAATAAGGAGGA J01749 86–108 AAAAAAAAATGAAATCTAACA ATGCGCTCAT-3′16 5′-GCTACATTTGAAGAGATA (SEQ ID NO: 27) ori15A X06403 1461-1397AATTGCACTGGATGCTAGAAA TTTTATCTGATTAATAAGATG ATC-3′ 175′-CGGAGATTTCCTGGAAGA (SEQ ID NO: 28) ″ ″ 780-829 TGCCTAGGAGATACTTAACAGGGAAGTGAGAG-3′ 18 5′-GTCTGCCGGATTGCTTAT (SEQ ID NO: 29) ori101 X016544490–4550 CCTGGCGGATCCGGTTGACAG TAAGACGGGTAAGCCTGTTGA T-3′ 195′-CCTAGGTTTCACCTGTTC (SEQ ID NO: 30) ″ ″ 6464-6408TATTAGGTGTTACATGCTGTT CATCTGTTACATTGTCGATCT G-3′ 205′-AGGCTTAAGTAGCACCCT (SEQ ID NO: 31) par X01654 4918-4858CGCAAGATCTGGGAAATCGCT GAATATTCCTTTTGTCTCCGA C-3′ 215′-GAGGGCGCCCCAGCTGGC (SEQ ID NO: 32) aphA2- V00618, 38-16,AATTCTAGACTCGAGCACTTT parA X04268 1–37 TGTTACCCGCGAAACAAAACCCAAAAACAAC-3′ 22 5′-AGAAGAAAAATCGAATTC (SEQ ID NO: 33) ″ X042681704-1644 GAGCATGAAGAGTTTCAGAAA ATGAGAGAGCGTGAGCAAGTG C-3′ 235′-CGAAGCCCAACCTTTCAT (SEQ ID NO: 34) ″ V00618 1044-986AGAAACTAGTGGTGGAATCGA AATCTCGTGATGGCAGGTT G-3′ 24 5′-GTTGTTTTTGGGTTTTGT(SEQ ID NO: 35) ″ X04268, 37-1, TTGGCGGGTAACAAAAGTGCT V00618 16–38CGAGTCTAGAATTGCCAGCTG GGGCGCCCTC-3′ ¹Relevant restriction sites areunderlined and referred to in the text; ribosome binding sites and startcodons are designated in italics. ²Refers to the sequence within thecoding strand of a given gene, to which the primer is homologous.³Refers to the sequence within the non-coding strand of a given gene, towhich the primer is homologous.

pTETnir15 (see Table 8; Oxer et al. 1991) was re-engineered such thatthe oriE1 origin of replication and bla gene were separated by a uniqueSpe1 site. Toward this end, an oriE1 cassette was synthesized by PCRusing Vent polymerase with primers 1 and 2 and pCVD315 (Galen et al.1990) as the template. The resulting 735 bp fragment carries engineeredSpe1 and BgllI sites 5′-proximal to the promoter controllingtranscription of RNA II, and an engineered AvrII site 675 bases fromthese sites. A separate PCR reaction was carried out using primers 3 and4 to create a 1234 bp bla cassette containing an engineered XbaI site5′-proximal to the original EcoRI site. The products from these two PCRreactions were gel purified and used in an overlapping PCR with primers1 and 4 to yield a final 1916 bp oriE1-bla fragment which wasself-ligated to create pJN1. The _(Pnir15)-tOXC fragment from pTETnir15was excised as an Eco RI (partial digestion)-Ava1 fragment, in which theAva1 terminus was polished, and inserted into the multiple cloningregion from pSL1180 (Brosius, 1989) cleaved with Eco RI and StuI; thiscassette was then re-excised as an Eco RI (partial digestion)—Avri1fragment and inserted into pJN1 cleaved with Eco RI-AvrII, creating pJN2(see Table 8).

6.9.2 Construction of pGFPompC

To facilitate screening of a functional osmotically regulated P_(ompC)allele from Escherichia coli, an aphA-2 cassette was constructed,encoding resistance to the aminoglycosides neomycin and kanamycin (Shawet al. 1993). A polymerase chain reaction (PCR) was carried out usingprimers 5 and 6 with the template pIB279 (Blomfield et al. 1991) togenerate a 1044 bp product, from which a promoterless 903 bp aphA-2BglII-NheI fragment was cleaved for replacement of a BglII-NheI toxCcassette encoding fragment C of tetanus toxin in pTETnir15. Theanaerobically regulated _(Pnir15) promoter was replaced with a 459 bpEcoRI-BglII P_(ompC) allele constructed using primers 7 and 8 withchromosomal template DNA from E. coli DH5α to create pKompC. Afterconfirming osmotic induction of P_(ompC) by examining the increase inresistance to kanamycin with increasing osmolarity, the aphA-2 cassettewas then replaced with a gfpuv gene encoding a prokaryoticcodon-optimized GFPuv allele (Clontech; Crameri et al. 1996). The gfpuvgene was recovered by PCR using primers 9 and 10 with the templatepGFPuv to generate a 751 bp BglII-NheI fragment which was inserted intopKompC, to generate pGFPompC. Colonies were screened for functionalGFPuv, and the brightest colonies were then examined for induction offluorescence with increasing concentrations of NaCl. A _(ompC1)-gfpuvcassette was cleaved from pGFPompC1 as an EcoRI-NheI fragment andinserted into a derivative of pJN2 cleaved with EcoRI-NheI to createpJJ4.

6.9.3 Construction of pNRB1, pGEN2, pGEN3, and pGEN4

Since it was intended that copy number not be influenced bytranscription originating from promoters outside the origin ofreplication, it was necessary to ensure that all replication cassetteswere flanked at both ends by transcription terminators. Because theorigin and antigen cassettes of pJN2 are separated by thetrpAterminator, it was only necessary to insert one additionalterminator between the origin and bla cassettes.

To facilitate construction of additional plasmids later on, a tetA-T1T2cassette was created. pYA292 (Galan et al. 1990) was first cleaved withHindIII and BglII, and the T1T2 terminator fragment was polished andinserted into the SmaI site of the pBluescript II KS (Stratagene)mutiple cloning region; when the proper orientation was identified, thiscassette was re-excised as a BamHI-PstI fragment and inserted intopIB307 (Blomfield et al. 1991) cleaved with BamHI-PstI, creating pJG14.It was later determined by sequence analysis that the cassette hadundergone a deletion of approximately 100 bp, removing half of the T2terminator.

Using pBR322 as a template, primers 11 and 12 were used to synthesize a1291 bp tetA BglII fragment. This tetA BgllI fragment was then insertedinto the BamHI site of pJG14 such that transcription of the tetA gene isterminated at the T1T2 terminator, creating pJG14tetA. Finally, thistetA-T1T2 cassette was cleaved from pJG14tetA as an EcoRI-PstI fragmentin which the PstI site had been removed by polishing; the resultingfragment was inserted into pJJ4, cleaved with SpeI, polished, andrecleaved with EcoRI to replace the bla cassette and create pNRB1.

The non-catalytic post-segregational killing function to be incorporatedinto the plasmid maintenance systems of the expression plasmidsdescribed here was the hok-sok locus, from the multiple drug resistanceR-factor pR1. Initial attempts at recovering the hok-sok locus after PCRwere unsuccessful. It was therefore necessary to use overlapping PCR togenerate a cassette in which hok-sok was transcriptionally fused to apromoterless teta gene such that transcription originating from the hokpromoter would continue into teta and result in a transcript encodingboth Hok and resistance to tetracycline. pR1 plasmid DNA was purifiedfrom E. coli J53(pR1) in which pRI encodes resistance to bothcarbenicillin and chloramphenicol. A 640 bp hok-sok fragment wassynthesized using primers 13 and 14; a promoterless 1245 bp tetafragment was recovered in a separate PCR using primers 15 and 12 withpNRB1 as the template. The products from these two PCR reactions werethen used in an overlapping PCR with primers 12 and 13 to yield thefinal 1816 bp hok-sok-tetA fragment. This fragment was inserted as anEcoRI-SphI fragment into pNRB1 cleaved with EcoRI-SphI, regenerating theteta gene and creating pGEN1.

A set of 3 isogenic plasmids was then constructed, differing only incopy number, from which all further expression plasmids would bederived. The BglII-AvrII origin of replication cassette of pGEN1 wasreplaced by a BglII-AvrII oriE1 cassette from pJN2 to generate pGEN2. Anori15A replication cassette was synthesized by PCR using primers 16 and17 with pACYC184 template to generate a 629 bp BamHI-AvrII fragment,which was inserted into pGEN2 cleaved with BgllI-AvrII to create pGEN3.Finally, an ori101 replication cassette was synthesized by PCR usingprimers 18 and 19 with pSC101 template, generating a 1949 bp BamHI-AvrIIfragment which was inserted into pGEN2 cleaved with BglII-AvrII tocreate pGEN4.

6.9.4 Construction of pJN5, pGEN51, pGEN91, and pGEN132

The principle set of isogenic expression plasmids, to which individualelements of a plasmid maintenance system were sequentially added, wascomposed of pGEN51 (containing oriE1), pGEN91 (containing ori15A), andpGEN132 (containing ori101). The basic replicon from which these 3plasmids were constructed was pJN5, which was assembled by cleaving theP_(ompC)-gfpuv cartridge as an EcoRI-NheI fragment from pGFPompC toreplace the _(Pnir15)-toxC cassette of pJN2. Construction of pGEN51 wasthen accomplished by removal of the replication cassette from pGEN2 as aBamHI fragment, and replacement of the origin of replication within pJN5digested with BglII and BamHI, thereby regenerating the gfpuv gene.Construction of pGEN91 and pGEN132 were constructed in an identicalmanner by excision of origin cassettes as BamHI fragments from pGEN3 andpGEN4 respectively (see FIG. 7 for representation of isogenic expressionplasmids based on pGEN91).

6.9.5 Construction of pJN6, pGEN71, pGEN111, and pGEN142

The hok-sok locus was then inserted as an XbaI-SaII fragment into pJN5cleaved with XbaI and SaII, again regenerating the gfpuv gene to createpJN6 (see Table 2). Construction of pGEN71, pGEN111, and pGEN142 wasthen carried out exactly as for pGEN51, pGEN91, and pGEN132 by insertioninto pJN6 of origin cassettes as BamHI fragments from pGEN 2, pGEN3, andpGEN4 respectively.

6.9.6 Construction of pJN7, pGEN84, and pGEN121

Construction of oriE1 and ori15A expression plasmids containing aplasmid maintenance system, composed of both a post-segregationalkilling system and at least one partition function, was first attemptedusing thepar function from pSC101. A 377 bp BamHI-BglII fragment wassynthesized using primers 18 and 20 with pSC101 template DNA; thisfragment was inserted into pJN6 cleaved with BglII to create pJN7. As inthe constructions above, origin cassettes from pGEN2 and pGEN3 were thenexcised as BamHI fragments and inserted into pJN7 digested with BgllIand BamHI to create pGEN84 and pGEN121.

6.9.7 Construction of pJN8, pGEN183, pGEN193, pGEN206, pGEN211 andpGEN222

The final expression plasmids were constructed by introduction of theparA active partitioning locus from pR1. As with hok-sok, initialattempts at recovering the parA locus after PCR were unsuccessful. Itwas necessary to use overlapping PCR to generate an aph-paraA cassette,in which aph and parA were divergently transcribed and separated by XbaI and XhoI sites, to enable subcloning of the parA locus. A 1737 bp parAfragment was synthesized using primers 21 and 22 with pR1 template; a1076 bp aphA-2 fragment was recovered in a separate PCR using primers 23and 24 with pIB279 as the template. The products from these two PCRreactions were then used in an overlapping PCR with primers 22 and 23 toyield the final 2743 bp aphA2-parA fragment. This fragment was insertedas a 2703 EcoRI-SpeI fragment into pJN6. The parA cassette was thenre-excised as an XhoI fragment and inserted again into pJN6 cleaved withXhoI, regenerating the gfpuv gene, and creating pJN8.

Plasmids carrying a plasmid maintenance system composed of thepost-segregational killing hok-sok function and parA, were constructedby excision of oriE1 and ori15A BamHI-SpeI cassettes from pGEN51 andpGEN91 respectively, and insertion into pJN8 cleaved with BamHI and SpeIto create pGEN183 and pGEN193 respectively. Plasmids containing the fullcomplement of hok-sok, par, and parA maintenance functions wereconstructed by insertion ofpar-containing origin cassettes as BamHI-SpeIcassettes from pGEN84, pGEN121, and pGEN132 into pJN8 cleaved with BamHIand SpeI to create pGEN211, pGEN222, and pGEN206 respectively.

6.10 Quantitation of GFPuv and Plasmid Maintenance

Quantitation of GFPuv and plasmid maintenance were analyzed by measuringthe fluorescence of plasmid-bearing live vectors using an Epics EliteESP flow cytometer/cell sorter system (Coulter) with the argon laserexciting bacteria at 488 nm and emissions detected at 525 nm. 25 ml 1×LBcultures grown as described above were pelleted, and bacteria wereresuspended into 1 ml of PBS. Cells were then diluted 1:1000 into PBSprior to determination of viable counts and flow analysis. Forwardversus side light scatter, measured with logarithmic amplifiers, wasused to gate on bacteria. A minimum of 50,000 events were acquired fromeach sample at a collection rate of approximately 3500 events persecond. Mean fluorescence intensity for a given bacterial population wasdetermined using the Epics Elite Software Analysis Package. The levelsof autofluorescence, determined using plasmidless S. typhi CVD 908-htrAand E. coli DH5α strains, were used to place markers quantitating thepercentages of bacteria in a given population expressing GFPuv.

6.11 Conclusions

The broad objective of the research presented in Sections 6.6–6.10 wasto investigate the feasibility of developing a plasmid maintenancesystem for the stabilization of multicopy expression plasmids encodingforeign antigens in an S. typhi live vector vaccine strain, withoutadditional modification of the chromosome. The maintenance of expressionplasmids was enhanced at two independent levels. First, dependence uponbalanced-lethal maintenance systems that involve catalytic enzymesexpressed from multicopy plasmids was removed; this was accomplishedthrough incorporation into expression plasmids of a post-segregationalkilling system based on the non-catalytic hok-sok plasmid addictionsystem from the antibiotic-resistance factor pR1. At least one naturallyoccurring plasmid partition function was also introduced into theseexpression plasmids, to potentially eliminate random segregation of suchplasmids, thereby enhancing their inheritance and stability.

Although these expression plasmids are ultimately intended to expressimmunogenic and protective antigens for delivery to the human immunesystem, GFPuv was selected as a test reporter antigen becausequantitation of mean fluorescence in a population of growing livevectors could be used as a measure of the stability of resident plasmidswithin the live vector. All expression plasmids carried an identicalantigen expression cassette, with a P_(ompC1) allele controllingtranscription, and translation optimized by incorporation of a consensusribosome binding site. Because no catalytic activity is associated withthe fluorescence of GFPuv, the level of fluorescence intensity measuredby flow cytometry within individual bacteria could be correlateddirectly with gene dosage and copy number. In addition, use of anosmotically regulated ompC promoter allowed an assessment of plasmidstability and live vector viability as increasing osmolarity inducedhigher levels of GFPuv synthesis and presumably higher levels ofmetabolic stress on the live vector. As seen in Table 2, we confirmedthat the P_(ompC1) allele engineered for these studies was responsive toincreased osmolarity; when driving expression of an aph-2 resistancegene, resistance to less than 50 μg/ml kanamycin was observed in theabsence of osmotic pressure but resistance increased to greater than 800μg/ml in the presence of 300 mM NaCl. It was surprising that althoughthe P_(ompC1) allele was engineered from the chromosomal locus of E.coli, it appeared to function more efficiently in S. typhi. Theuninduced level of expression of GFPuv was the same for both DH5α andCVD 908-htrA (mean fluorescence intensity of 4.45 vs 5.37 respectively,Table 3). However, GFPuv synthesis increased 70% in DH5α afterinduction, but rose over 300% in CVD 908-htrA (mean fluorescenceintensity of 7.69 vs 23.4 respectively). This effect was not limited tothe P_(ompC1) allele but was equally remarkable when using P_(ompC3)(Table 3). These data do not agree with recent observations ofMartinez-Flores et al (1999) who reported that E. coli ompC-lacZ geneticfusions expressed constitutively within S. typhi, and that thisconstitutive level of expression was comparable to induced levels withinE. coli. Although we have identified a defined locus of point mutationsat the 3′-terminus of our E. coli P_(ompC1) allele which could explainits osmotically controlled behavior within S. typhi CVD 908-htrA, suchmutations were not identified within P_(ompC3), which also responds toosmolarity within CVD 908-htrA. It should be noted, however, that thegenetic fusions studied by Martinez-Flores et al involved 1,150 bp ofthe E. coli 5′ ompC upstream control region, while the P_(ompC) allelesconstructed here involve only 459 bp of the 5′-proximal control regionof ompC. Regardless of this discrepancy, it is encouraging that thehighest levels of regulated heterologous gene expression are observedwithin the attenuated S. typhi live vector vaccine strain.

The contributions of several plasmid maintenance systems to thestability of plasmids within CVD 908-htrA, growing in the absence ofantibiotic selection, were then examined. No combination of maintenancefunctions could stabilize plasmids containing oriE1 origins ofreplication; in fact, these constructs were difficult to propagate evenin the presence of antibiotic. These observations cast doubt upon therationale for using higher copy number plasmids to optimize expressionof heterologous antigens within the cytoplasm of S. typhi-based livevectors, a strategy that, heretofore, has been followed by other groupsinvestigating Salmonella as live vectors (Covone et al. 1998).

Incorporation of plasmid maintenance systems into plasmids carrying anori15A origin of replication was more encouraging. When live vectorscarrying such plasmids were passage without selection for 24 hr at 37°C., the effects of various combinations of maintenance functions becameapparent. In the absence of maintenance functions, the ori15A repliconpGEN91 was lost from greater than 90% of the population, regardless ofthe level of induction of P_(ompC1) (see Table 6 and FIG. 8). Withincorporation of the hok-sok post-segregational killing locus inpGEN111, the percentage of bacteria expressing GFPuv tripled under allinduction conditions, confirming the observations of others that thehok-sok locus enhances the stability of ori15A replicons (Gerdes et al.1985; Gerdes, 1988; Gerdes et al. 1997b). However, it was still notedthat regardless of induction conditions, greater than 50% of thebacterial population no longer fluoresced. Since it was confirmed thatat least a portion of this non-fluorescing population was still viableand lacked drug resistance, these data confirm previous reports (Gerdeset al. 1986; Wu and Wood, 1994; Pecota et al. 1997) that the presence ofa hok-sok post-segregational killing system is insufficient by itself toensure that plasmidless viable bacteria will not arise in a growingpopulation.

One possible mechanism that allows for escape from the influence ofhok-sok involves spontaneous point mutations arising within the lethalHok open reading frame, which could conformationally inactivate Hok andthereby allow plasmid loss to occur without lethality. This pointemphasizes the requirement of multiple mechanisms for enhancing thestability of resident plasmids within growing bacteria; should onemaintenance function become inactivated, the probability of otherindependent functions simultaneously becoming inactivated becomesvanishingly small. Indeed, such redundancy in maintenance functions iswidespread within naturally occurring low copy number plasmids(Nordstrom and Austin, 1989). For example, the Escherichia coli sexfactor F contains one active partitioning function (sop) and. twokilling systems (ccd and flm) (Loh et al. 1988; Golub and Panzer, 1988;Van Melderen et al. 1994; Niki and Hiraga, 1997). Similarly, the drugresistance plasmid pR1 contains the active partitioning functionparA, aswell as the post-segregational killing system hok-sok; in addition, itcarries yet another recently defined kis-kid killing system (Bravo etal. 1987; Bravo et al. 1988; Ruiz-Echevarria et al. 1995). Wedemonstrate in work reported here that insertion into multicopy ori15Areplicons of a more complete maintenance system, composed of both apost-segregational system and two partition fimctions, dramaticallyimproves the stability of these expression plasmids in the absence ofselection, regardless of induction conditions for heterologous antigenexpression. However, after passage without selection for 48 hrs,plasmids were eventually lost from the bacterial population, due toescape from the lethality of Hok. This problem has recently beenaddressed by Pecota et al (1997) who reported that incorporation of dualkilling systems significantly improved plasmid stability when comparedto the use of hok-sok alone; no partition functions were present inthese plasmids. Perhaps inclusion of the kis-kid killing system, to morefully represent the complement of pR1 stability functions, may berequired for optimal stability of higher copy expression plasmids withinS. typhi live vectors; since phd-doc PSK cassettes have recently beenconstructed, we are also examining the compatibility of this PSKfunction in our expression plasmids pGEN211, pGEN222 and pGEN206.

A comparison of strains carrying pGEN121 (an ori15A replicon carryinghok-sok+par, ˜15 copies per chromosomal equivalent) with the much lowercopy number plasmid pGEN142 (an ori101 replicon carrying hok-sok+par, 5copies per chromosomal equivalent) shows that under conditions ofmaximum induction of P_(ompC1) with 300 mM NaCl, 57% of a population ofCVD 908-htrA(pGEN121), passaged for only 24 hr without selection,fluoresce with a mean fluorescence intensity of 105.3; for a populationof CVD908-htrA(pGEN142), passaged for 96 hr without selection underidentical induction conditions, 94% of the bacteria analyzed by flowcytometry still maintain a mean fluorescence intensity of 47.7. Based onsuch results with GFPuv as a test antigen, it is tempting to speculatethat an optimum level of heterologous antigen presented by an attenuatedS. typhi-based live vector vaccine to the human immune system can beachieved by decreasing the copy number of resident expression plasmidsto perhaps 5 copies per chromosomal equivalent.

The efficiency of eliciting an immune response directed against aheterologous antigen will depend in part upon the ability of the livevector to present such antigens to the immune system. The ability of alive vector to present antigens will in turn depend upon the stabilityof multicopy expression plasmids that encode the heterologous antigens.Our results demonstrate that inclusion of a plasmid maintenance systemwithin multicopy expression plasmids, without further geneticmanipulation of the live vector, enhances the stability of suchexpression plasmids. However, the presence of multicopy plasmids mayalso influence the metabolic fitness of the live vector. This isrelevant because some foreign antigens of interest exert a deleteriouseffect on the live vector.

While we do not intend to be bound to this theory, we conclude that asignificant metabolic burden is placed upon CVD 908-htrA carrying amulticopy expression plasmid; as copy number and/or level of geneexpression increases, metabolic burden increases. Studies with E. Colihave clearly established that plasmid-bearing bacteria grow slower thanplasmidless bacteria (Boe et al. 1987; McDermott et al. 1993; Wu andWood, 1994; Pecota et al. 1997; Summers, 1998). It has also beendemonstrated that as copy number increases, the growth rate of suchstrains decreases; similarly, as induction of heterologous genesincreases, growth rate decreases further (Wu and Wood, 1994; Pecota etal. 1997). Clearly, spontaneous plasmid loss would remove any metabolicburden and allow plasmidless bacteria to quickly outgrow the populationof plasmid-bearing bacteria. In elegant studies, Wu and Wood (Wu andWood, 1994) showed that plasmid-bearing E. coli strains maintainedplasmids under conditions where cloned gene expression was low for 100hr when passaged in the absence of selection; in contrast, under maximuminduction conditions, complete plasmid loss occurred within 10 hr.Interestingly, when the hok-sok locus was inserted into these expressionplasmids, the plasmids were maintained for 300 hr. under uninducedconditions and 30 hr. under inducing conditions. Such a shift in antigenexpression within a population of live vector bacteria would be expectedto reduce the efficiency of stimulating any immune response specific tothe foreign antigen. Our analysis leads us to conclude that the goal foran effective multivalent S. typhi-based live vector vaccine is tooptimize viability using stabilized lower copy number expressionvectors, capable of expressing high levels of heterologous antigen inresponse to an environmental signal likely to be encountered in vivoafter the vaccine organisms have reached an appropriate ecologicalniche. We are currently testing this strategy using the murineintranasal model to examine the immunogenicity of fragment C of tetanustoxin expressed within CVD 908-htrA from our expression vectors pGEN211(oriE1), pGEN222 (ori15A), and pGEN206 (ori101), all of which carryidentical plasmid maintenance systems and differ only in copy number.The work presented herein enables the development of single dose, oralS. typhi-based live vector vaccines capable of inducing protectiveimmune responses against multiple unrelated human pathogens.

7. References

The disclosures of the following references are incorporated herein intheir entirety:

Acheson, D. W. K. 1998. Nomenclature of enterotoxins. Lancet351:1003.

Acheson, D. W. K., M. M. Levine, J. B. Kaper, and G. T. Keusch. 1996.Protective immunity to Shiga-like toxin I following oral immunizationwith Shiga-like toxin I B-subunit-producing Vibrio cholerae CVD 103-HgR.Infection and Immunity 64:355.

Austin, S. J. 1988. Plasmid partition. Plasmid 20:1.

Austin, S., S. Friedman, and D. Ludtke. 1986. Partition functions ofunit-copy plasmids can stabilize the maintenance of plasmid pBR322 atlow copy number. J Bacteriol 168: 1010–1013.

Barry, E. M., O. G. Gomez-Duarte, S. Chatfield, R. Rappuoli, M. Pizza,G. Losonsky, J. E. Galen, and M. M. Levine. 1996. Expression andimmunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment Cfusions in Salmonella typhi vaccine strain CVD 908. Infection andImmunity 64:4172–4181

Barth, P. T., H. Richards, and N. Datta. 1978. Copy numbers ofcoexisting plasmids in Escherichia coli K-1 2. J Bacteriol 135: 760–765.

Bast, D. J., J. L. Brunt n, M. A. Karmali, and S. E. Richardson. 1997.Toxicity and immunogenicity of a verotoxin 1 mutant with reducedglobotriaosylceramide receptor binding in rabbits. Infection andImmunity 65:2019.

Baumler, A. J., J. G. Kusters, I. Stojiljkovic, and F. Heffron. 1994.Salmonella typhimurium loci involved in survival within macrophages.Infection and Immunity 62:1623.

Beaucage, S. L., C. A. Miller, and S. N. Cohen. 1991. Gyrase-dependentstabilization of pSC101 plasmid inheritance by transcriptionally activepromoters. EMBO J 10: 2583–2588.

Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland,M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J.Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B.Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coliK-12. Science 277:1453.

Blomfield, I. C., V. Vaughn, R. F. Rest, and B. I. Eisenstein. 1991.Allelic exchange in Escherichia coli using the Bacillus subtilis sacBgene and a temperature-sensitive pSC101 replicon. Molecular Microbiology5:1447–1457.

Boe, L. and K. V. Rasmussen. 1996. Suggestions as to quantitativemeasurements of plasmid loss. Plasmid 36:153.

Boe, L., K. Gerdes, and S. Molin. 1987. Effects of genes exerting growthinhibition and plasmid stability on plasmid maintenance. Journal ofBacteriology 169:4646–4650.

Bokman, S. H. and W. W. Ward. 1981. Renaturation of Aequoreagreen-fluorescent protein. Biochemical and Biophysical ResearchCommunications 101:1372.

Bosworth, B. T., J. E. Samuel, H. W. Moon, A. D. O'Brien, V. M. Gordon,and S. C. Whipp. 1996. Vaccination with genetically modified Shiga-liketoxin IIe prevents edema disease in swine. Infection and Immunity 64:55.

Bouvier, J., C. Richaud, W. Higgins, O. Bogler, and P. Stragier. 1992.Cloning, characterization, and expression of the dapE gene ofEscherichia coli. Journal of Bacteriology 174:5265.

Boyd, B. and C. A. Lingwood. 1989. Verotoxin receptor glycolipid inhuman renal tissue. Nephron 51:207.

Bravo, A., G. de Torrontegui, and R. Diaz. 1987. Identification ofcomponents of a new stability system of plasmid R1, ParD, that is closeto the origin of replication of this plasmid. Mol Gen Genet 210:101–110.

Brayo, A., S. Ortega, G. de Torrontegui, and R. Diaz. 1988. Killing ofEscherichia coli cells modulated by components of the stability systemparD of plasmid R1. Mol Gen Genet 215: 146–151.

Brosius, J. 1989. Superpolylinkers in cloning and expression vectors.DNA 8: 759–777.

Butterton, J. R., E. T. Ryan, D. W. Acheson, and S. B. Calderwood. 1997.Coexpression of the B subunit of Shiga toxin 1 and EaeA fromenterohemorrhagic Escherichia coli in Vibrio cholerae vaccine strains.Infection and Immunity 65:2127–2135

Cabello, F., K. Timmis, and S. N. Cohen. 1976. Replication control in acomposite plasmid constructed by in vitro linkage of two distinctreplicons. Nature 259: 285–290.

Calderwood, S. B., D. W. K. Acheson, G. T. Keusch, T. J. Barrett, P. M.Griffin, N. A. Strockbine, B. Swaminathan, J. B. Kaper, M. M. Levine, B.S. Kaplan, H. Karch, A. D. O'Brien, T. G. Obrig, Y. Takeda, P. I. Tarr,and I. K. Wachsmuth. 1996. Proposed new nomenclature for SLT (VT)family. ASM News 62:118.

Calderwood, S. B., F. Auclair, A. Donohue-Rolfe, G. T. Keusch, and J. J.Mekalanos. 1987. Nucleotide sequence of the Shiga-like toxin genes ofEscherichia coli. Proceedings of the National Academy of Sciences USA84:4364.

Carlini, L. E., R. D. P rter, U. Curth, and C. Urbanke. 1993. Viabilityand preliminary in vivo characterization of site-specific mutants ofEscherichia coli single-stranded DNA-binding protein. MolecularMicrobiology 10:1067.

Carter, P. B. and F. M. Collins. 1974. Growth of typhoid and paratyphoidbacilli in intravenously infected mice. Infection and Immunity 10:816.

Cerin, H. and J. Hackett. 1989. Molecular cloning and analysis of theincompatibility and partition functions of the virulence plasmid ofSalmonella typhimurium. Microbial Pathogenesis 7:85.

Cerin, H. and J. Hackett. 1993. The parVP region of the Salmonellatyphimurium virulence plasmid pSLT contains four loci required forincompatibility and partition. Plasmid 30:30.

Chalfie, M., Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher. 1994.Green fluorescent protein as a marker for gene expression. Science263:802.

Chambers, S. P., S. E. Prior, D. A. Barstow, and N. P. Minton. 1988. ThepMTLnic cloning vectors. I. Improved pUC polylinker regions tofacilitate the use of sonicated DNA for nucleotide sequencing. Gene68:139.

Chang, A. C. Y., and S. N. Cohen. 1978. Construction andcharacterization of amplifiable multicopy DNA cloning vehicles derivedfrom the P15A cryptic miniplasmid. J Bacteriol 134: 1141–1156.

Chase, J. W. and K. R. Williams. 1986. Single-stranded DNA bindingproteins required for DNA replication. Annual Reviews in Biochemistry55:103.

Chase, J. W., J. B. Murphy, R. F. Whittier, E. Lorensen, and J. J.Sninsky. 1983. Amplification of ssb-1 mutant single-stranded DNA-bindingprotein in Escherichia coli. Journal of Molecular Biology 163,164:193.

Chatfield, S., K. Strahan, D. Pickard, I. G. Charles, C. E. Hormaeche,and G. Dougan. 1992. Evaluation of Salmonella typhimurium strainsharbouring defined mutations in htrA and aroA in the murinesalmonellosis model. Microbial Pathogenesis 12:145.

Clark, C., D. Bast, A. M. Sharp, P. M. St. Hilaire, R. Agha, P. E.Stein, E. J. Toone, R. J. Read, and J. L. Brunton. 1996. Phenylalanine30 plays an important role in receptor binding of verotoxin-1. MolecularMicrobiology 19 :891.

Conradi, H. 1903. Ueber losliche, durch aseptische autolyse erhaltenegiftstoffe von ruhr-und Typhusbazillen. Dtsch. Med. Wochenschr. 29:26.

Covarrubias, L., L. Cervantes, A. Covarrubias, X. Soberon, I. Vichido,A. Blanco, Y. M. Kupersztoch-Portnoy, and F. Bolivar. 1981. Constructionand characterization of new cloning vehicles. V. Mobilization and codingproperties of pBR322 and several deletion derivatives including pBR327and pBR328. Gene 13: 25–35.

Covone, M. G., M. Brocchi, E. Palla, W. D. da Silveira, R. Rappuoli, andC. L. Galeotti. 1998. Levels of expression and immunogenicity ofattenuated Salmonella enterica serovar typhimurium strains expressingEscherichia coli mutant heat-labile enterotoxin. Infection and Immunity66:224–231

Crameri, A., E. A. Whitehorn, E. Tate, and W. P. Stemmer. 1996. Improvedgreen fluorescent protein by molecular evolution using DNA shuffling.Nat Biotechnol 14: 315–319.

Dam, M. and K. Gerdes. 1994. Partitioning of plasmid R1: ten directrepeats flanking the parA promoter constitute a centromere-likepartition site parC, that expresses incompatibility. Journal ofMolecular Biology 236:1289–1298.

Dopf, J. and T. M. Horiagon. 1996. Deletion mapping of the Aequoreavictoria green fluorescent protein. Gene 173:39.

Downes, F. P., T. J. Barrett, J. H. Green, C. H. Aloisio, J. S. Spika,N. A. Strockbine, and I. K. Wachsmuth. 1988. Affinity purification andcharacterization of Shiga-like toxin II and production of toxin-specificmonoclonal antibodies. Infection and Immunity 56:1926.

Egger, L. A., H. Park, and M. Inouye. 1997. Signal transduction via thehistidyl-aspartyl phosphorelay. Genes to Cells 2:167.

Endo, Y., K. Tsurugi, T. Yutsudo, Y. Takeda, T. Ogasawara, and K.Igarashi. 1988. Site of action of a Vero toxin (VT2) from Escherichiacoli O157:H7 and of Shiga toxin on eukaryotic ribosomes: RNAN-glycosidase activity of the toxins. European Journal of Biochemistry171:45.

Forrest, B. D., J. T. Labrooy, S. R. Attridge, G. Boehm, L. Beyer, R.Morona, D. J. C. Shearman, and D. Rowley. 1989. Immunogenicity of acandidate live oral typhoid/cholera hybrid vaccine in humans. J. Infect.Dis. 159: 145.

Fraser, M. E., M. M. Chernaia, Y. V. Kozlov, and M. N. G. James. 1994.Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 Aresolution. Nature Structural Biology 1:59.

Galan, J. E., K. Nakayama, and R. Curtiss III. 1990. Cloning andcharacterization of the asd gene of Salmonella typhimurium: use instable maintenance of recombinant plasmids in Salmonella vaccinestrains. Gene 94:29–35.

Galen, J. E. and M. M. Levine. 1995. Improved suicide vectors forchromosomal mutagenesis in Salmonella typhi. Abstracts of the AnnualMeeting of the American Society of Microbiology H192:(Abstract)

Galen, J. E. and M. M. Levine. 1996. Further refinements of suicidevector-mediated chromosomal mutagenesis in Salmonella typhi. Abstractsof the Annual Meeting of the American Society of MicrobiologyH260:(Abstract)

Galen, J. E., O. G. Gomez-Duarte, G. Losonsky, J. L. Halpern, C. S.Lauderbaugh, S. Kaintuck, M. K. Reymann, and M. M. Levine. 1997. Amurine model of intranasal immunization to assess the immunogenicity ofattenuated Salmonella typhi live vector vaccines in stimulating serumantibody responses to expressed foreign antigens. Vaccine 15:700–708.

Galen, J. E., E. R. Vimr, L. Lawrisuk, and J. B. Kaper. 1990. Cloning,sequencing, and expression of the gene, nanH, for Vibrio choleraeneuraminidase. In Advances in research on cholera and related diarrheas(Edited by Sack R. B. and Zinnake Y. Tokyo: KTK Scientific Publishers.pp. 143–153.

Gay, P., D. Le Coq, M. Steinmetz, E. Ferrari, and J. A. Hoch. 1983.Cloning structural gene sacB, which codes for exoenzyme levansucrase ofBacillus subtilis: expression of the gene in Escherichia coli. Journalof Bacteriology 153:1424.

Gerdes, K. 1988. The parB (hok-sok) locus of plasmid R1: a generalpurpose plasmid stabilization system. Bio/Technology 6: 1402–1405.

Gerdes, K. and S. Molin. 1986. Partitioning of plasmid R1: structuraland functional analysis of the parA locus. Journal of Molecular Biology190:269.

Gerdes, K., A. P. Gultyaev, T. Franch, K. Pedersen, and N. D. Mikkelsen.1997. Antisense RNA–regulated programmed cell death. Annual Reviews inGenetics 31:1-31.

Gerdes, K., J. S. Jacobsen, and T. Franch. 1997b. Plasmid stabilizationby post-segregational killing. Genet Eng (NY) 19: 49–61.

Gerdes, K., J. E. Larsen, and S. Molin. 1985. Stable inheritance ofplasmid R1 requires two different loci. J Bacteriol 161: 292–298.

Gerdes, K., P. B. Rasmussen, and S. Molin. 1986. Unique type of plasmidmaintenance function: postsegregational killing of plasmid-free cells.Proc Natl Acad Sci USA 83: 3116–3120.

Gerichter, C. B. 1960. The dissemination of Salmonella typhi, S.paratyphi A, and S. paratyphi B through the organs of the white mouse byoral infection. Journal of Hygiene, Cambridge 58:307.

Gerichter, C. B. and D. L. Boros. 1962. Dynamics of infection of theblood stream and internal organs of white mice with Salmonella typhi byintraperitoneal injection. Journal of Hygiene, Cambridge 60:311.

Golub, E. I., and H. A. Panzer. 1988. The F factor of Escherichia colicarries a locus of stable plasmid inheritance stm, similar to the parBlocus of plasmid R1. Mol Gen Genet 214: 353–357.

Gomez-Duarte, O. G., J. E. Galen, S. N. Chatfield, R. Rappuoli, L.Eidels, and M. M. Levine. 1995. Expression of fragment C of tetanustoxin fused to a carboxyl-terminal fragment of diphtheria toxin inSalmonella typhi CVD 908 vaccine strain. Vaccine 13:1596.

Gonzalez, C., D. M. Hone, F. Noriega, C. O. Tacket, J. R. Davis, G.Losonsky, J. P. Nataro, S. Hoffman, A. Malik, E. Nardin, M. Sztein, D.G. Heppner, T. R. Fouts, A. Isibasi, and M. M. Levine. 1994. Salmonellatyphi vaccine strain CVD 908 expressing the circumsporozoite protein ofPlasmodium falciparum: strain construction and safety and immunogenicityin humans. Journal of Infectious Diseases 169:927–931.

Gordon, V. M., S. C. Whipp, H. W. Moon, A. D. O'Brien, and J. E. Samuel.1992. An enzymatic mutant of Shiga-like toxin II variant is a vaccinecandidate for edema disease of swine. Infection and Immunity 60:485.

Gottesman, S., W. P. Clark, V. de Crecy-Lagard, and M. R. Maurizi. 1993.ClpX, an alternative subunit for the ATP-dependent Clp protease ofEscherichia coli. Journal of Biological Chemistry 268:22618.

Green, J. M., B. P. Nichols, and R. G. Matthews. 1996. Folatebiosynthesis, reduction, and polyglutamylation. In Escherichia coli andSalmonella: Cellular and molecular biology. 2nd ed. F. C. Neidhardt, R.Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter and H. E. Umbarger, eds. ASMPress, Washington, D.C. p. 665.

Griffin, P. M. 1995. Escherichia coli O157:H7 and otherenterohemorrhagic Escherichia coli. In Infections of thegastrointestinal tract. M. J. Blaser, P. D. Smith, J. I. Ravdin, H. B.Greenberg and R. L. Guerrant, eds. Raven Press, Ltd, New York, p. 739.

Gyles, C. L. 1992. Escherichia coli cytotoxins and enterotoxins.Canadian Journal of Microbiology 38:734.

Heim, R., D. C. Prasher, and R. Y. Tsien. 1994. Wavelength mutations andposttranscriptional autoxidation of green fluorescent protein.Proceedings of the National Academy of Sciences USA 91:12501.

Hiszczynska-Sawicka, E., and J. Kur. 1997. Effect of Escherichia coliIHF mutations on plasmid p15A copy number. Plasmid 38: 174–179.

Hoiseth, S. K. and B. A. Stocker. 1981. Aromatic-dependent Salmonellatyphimurium are non-virulent and effective as live vaccines. Nature291:238.

Hone, D. M., A. M. Harris, S. Chatfield, G. Dougan, and M. M. Levine.1991. Construction of genetically defined double aro mutants ofSalmonella typhi. Vaccine 9: 810–816.

Hovde, C. J., S. B. Calderwood, J. J. Mekalanos, and R. J. Collier.1988. Evidence that glutamic acid 167 is an active-site residue ofShiga-like toxin I. Proceedings of the National Academy of Sciences USA85:2568.

Jackson, M. P., E. A. Wadolkowski, D. L. Weinstein, R. K. Holmes, and A.D. O'Brien. 1990. Functional analysis of the Shiga toxin and Shiga-liketoxin type II variant binding subunits by using site-directedmutagenesis. Journal of Bacteriology 172:653.

Jackson, M. P., R. J. Neill, A. D. O'Brien, R. K. Holmes, and J. W.Newland. 1987. Nucleotide sequence analysis and comparison of thestructural genes for Shiga-like toxin I and Shiga-like toxin II encodedby bacteriophages from Escherichia coli. FEMS Microbiology Letters44:109.

Jackson, M. P., R. L. Deresiewicz, and S. B. Calderwood. 1990.Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymaticsubunits. Journal of Bacteriology 172:3346.

Jarvis, K. G. and J. B. Kaper. 1996. Secretion of extracellular proteinsby enterohemorrhagic Escherichia coli via a putative type III secretionsystem. Infection and Immunity 64:4826.

Jarvis, K. G., J. A. Giron, A. E. Jerse, T. K. McDaniel, M. S.Donnenberg, and J. B. Kaper. 1995. Enteropathogenic Escherichia colicontains a putative type III secretion system necessary for the exportof proteins involved in attaching and effacing lesion formation.Proceedings of the National Academy of Sciences USA 92:7996.

Jensen, R. B. and K. Gerdes. 1995. Programmed cell death in bacteria:proteic plasmid stabilization systems. Molecular Microbiology 17:205.

Jensen, R. B. and K. Gerdes. 1997. Partitioning of plasmid R1. The ParMprotein exhibits ATPase activity and interacts with the centromere-likeParR-parC complex. Journal of Molecular Biology 269:505–513.

Karem, K. L., S. Chatfield, N. Kuklin, and B. T. Rouse. 1995.Differential induction of carrier antigen-specific immunity bySalmonella typhimurium live-vaccine strains after single mucosal orintravenous immunization of BALB/c mice. Infection and Immunity63:4557–4563.

Karmali, M. A. 1989. Infection by verocytotoxin-producing Escherichiacoli. Clinical Microbiological Reviews 2:15.

Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, and B. T. Steele.1983. Escherichia coli cytotoxin, haemolytic-uraemic syndrome, andhaemorrhagic colitis. Lancet ii:1299.

Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus, and H.Lior. 1985. The association between idiopathic hemolytic uremic syndromeand infection by verotoxin-producing Escherichia coli. Journal ofInfectious Diseases 151:775.

Karpman, D., H. Connell, M. Svensson, F. Scheutz, P. Alm, and C.Svanborg. 1997. The role of lipopolysaccharide and Shiga-like toxin in amouse model of Escherichia coli O157:H7 infection. Journal of InfectiousDiseases 175:611.

Keusch, G. T., G. F. Grady, L. J. Mata, and J. McIver. 1972.Pathogenesis of shigella diarrhea. 1. Enterotoxin production by Shigelladysenteriae 1. Journal of Clinical Investigation 51:1212.

Killeen, K. P., V. Escuyer, J. J. Mekalanos, and R. J. Collier. 1992.Reversion of recombinant toxoids: mutations in diphtheria toxin thatpartially compensate for active-site deletions. Proceeding of theNational Academy of Sciences USA 89:6207.

Kim, J. Y., H. A. Kang, and D. D. Ryu. 1993. Effects of the par locus onthe growth rate and structural stability of recombinant cells.Biotechnology Progress 9:548.

Konowalchuk, J., J. I. Speirs, and S. Stavric. 1977. Vero response to acytotoxin of Escherichia coli. Infection and Immunity 18:775.

Langermann, S., S. Palaszynski, A. Sadziene, C. K. Stover, and S.Koenig. 1994. Systemic and mucosal immunity induced by BCG vectorexpressing outer-surface protein A of Borrelia burgdorferi. Nature 372:552–555.

Lee, S. F., R. J. March, S. A. Halpern, G. Faulkner, and L. Gao. 1999.Surface expression of a protective recombinant pertussis toxin S1subunit fragment in Streptococcus gordonii. Infect immun 67: 1511–1516.

Lehnherr, H. and M. B. Yarmolinsky. 1995. Addiction protein Phd ofplasmid prophage P1 is a substrate of the CIpXP serine protease ofEscherichia coli. Proceedings of the National Academy of Sciences USA92:3274.

Lehnherr, H., E. Maguin, S. Jafri, and M. B. Yarmolinsky. 1993. Plasmidaddiction genes of bacteriophage P1: doc, which causes cell death oncuring of prophage, and phd, which prevents host death when prophage isretained. Journal of Molecular Biology 233:414.

Levine, M. M., J. E. Galen, E. M. Barry, F. Noriega, S. Chatfield, M.Sztein, G. Dougan, and C. O. Tacket. 1996. Attenuated Salmonella as liveoral vaccines against typhoid fever and as live vectors. Journal ofBiotechnology 44:193.

Lindgren, S. W., J. E. Samuel, C. K. Schmitt, and A. D. O'Brien. 1994.The specific activities of Shiga-like toxin type II (SLT-II) andSLT-II-related toxins of enterohemorrhagic Escherichia coli differ whenmeasured by Vero cell cytotoxicity but not by mouse lethality. Infectionand Immunity 62:623.

Lloyd, R. G. and K. B. Low. 1996. Homologous recombination. InEscherichia coli and Salmonella: Cellular and molecular biology. 2nd ed.F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B.Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.Umbarger, eds. ASM Press, Washington, D.C. p. 2236.

Loh, S. M., D. S. Cram, and R. A. Skurray. 1988. Nucleotide sequence andtranscriptional analysis of a third function (Flm) involved in F plasmidmaintenance. Gene 66: 259–268.

Lohman, T. M. and M. E. Ferrari. 1994. Escherichia coli single-strandedDNA-binding protein: multiple DNA-binding modes and cooperativities.Annual Reviews in Biochemistry 63:527.

Louise, C. B. and T. G. Obrig. 1995. Specific interaction of Escherichiacoli O157:H7-derived Shiga-like toxin II with human renal endothelialcells. Journal of Infectious Diseases 172:1397.

Love, C. A., P. E. Lilley, and N. E. Dixon. 1996. Stablehigh-copy-number bacteriophage lambda promoter vectors foroverproduction of proteins in Escherichia coli. Gene 176:49.

Lynch, A. S. and E. C. C. Lin. 1996. Responses to molecular oxygen. InEscherichia coli and Salmonella: Cellular and molecular biology. 2nd ed.F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B.Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.Umbarger, eds. ASM Press, Washington, D.C. p. 1526.

Magnuson, R., H. Lehnherr, G. Mukhopadhyay, and M. B. Yarmolinsky. 1996.Autoregulation of the plasmid addiction operon of bacteriophage P1.Journal of Biological Chemistry 271:18705.

Makoff, A. J., and A. E. Smallwood. 1988. Heterologous expression inEscherichia coli: effects of alterations in the sequence 5′ to theinitiation codon. Biochem Soc Trans 16: 48–49.

Mangeney, M., C. A. Lingwood, S. Taga, B. Caillou, T. Tursz, and J.Wiels. 1993. Apoptosis induced in Burkitt's lymphoma cells viaGb.sub.3/CD77, a glycolipid antigen. Cancer Research 53:5314.

Marshall, J., R. Molloy, G. W. J. Moss, J. R. Howe, and T. E. Hughes.1995. The jellyfish green fluorescent protein: a new tool for studyingion channel expression and function. Neuron 14:211.

Martinez-Flores, I., R. Cano, V. H. Bustamante, E. Calva, and J. L.Puente. 1999. The ompB operon partially determines differentialexpression of OmpC in Salmonella typhi and Escherichia coli. J Bacteriol181: 556–562.

Matthews, R. G. 1996. One-carbon metabolism. In Escherichia coli andSalmonella: Cellular and molecular biology. 2nd ed. F. C. Neidhardt, R.Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter and H. E. Umbarger, eds. ASMPress, Washington, D.C. p. 600.

Maurizi, M. R., W. P. Clark, Y. Katayama, S. Rudikoff, J. Pumphrey, B.Bowers, and S. Gottesman. 1990. Sequence and structure of CIp P, theproteolytic component of the ATP-dependent CIp protease of Escherichiacoli. Journal of Biological Chemistry 265:12536.

McClelland, M. and. R. Wilson. 1998. Sample sequencing of the Salmonellatyphi genome: comparison to the E. coli K-12 genome. Infection andImmunity

McDaniel, T. K., K. G. Jarvis, M. S. Donnenberg, and J. B. Kaper. 1995.A genetic locus of enterocyte effacement conserved among diverseenterobacterial pathogens. Proceedings of the National Academy ofSciences USA 92:1664.

McDermott, P. J., P. Gowland, and P. C. Gowland. 1993. Adaptation ofEscherichia coli growth rates to the presence of pBR322. Lett ApplMicrobiol 17: 139–143.

Meacock, P. A., and S. N. Cohen. 1980. Partitioning of bacterialplasmids during cell division: a cis-acting locus that accomplishesstable plasmid inheritance. Cell 20: 529–542.

Medaglini, D., G. Pozzi, T. P. King, and V. A. Fischetti. 1995. Mucosaland systemic immune responses to a recombinant protein expressed on thesurface of the oral commensal bacterium Streptococcus gordonii afteroral colonization. Proc Natl Acad Sci USA 92: 6868–4872.

Melton-Celsa, A. R. and A. D. O'Brien. 1998. The structure, biology, andrelative toxicity for cells and animals of Shiga toxin family members.In Escherichia coli O157:H7 and other Shiga toxin-producing E. colistrains. J. B. Kaper and A. D. O'Brien, eds. ASM Press, Washington, D.C.In press.

Mikkelsen, N. D. and K. Gerdes. 1997. Sok antisense RNA from plasmid R1is functionally inactivated by RNaseE and polyadenylated by poly(A)polymerase I. Molecular Microbiology 26:311.

Miller, C. A., S. L. Beaucage, and S. N. Cohen. 1990. Role of DNAsuperhelicity in partitioning of the pSC101 plasmid. Cell 62: 127–133.

Moxley, R. A. and D. H. Francis. 1998. Overview of Animal Models. InEschenchia coli O157:H7 and other Shiga toxin-producing E. coli strains.J. B. Kaper and A. D. O'Brien, eds. ASM Press, Washington, D.C. Inpress.

Muhldorfer, I., J. Hacker, G. T. Keusch, D. W. Acheson, H. Tschape, A.V. Kane, A. Ritter, T. Olschlager, and A. Donohue-Rolfe. 1996.Regulation of the Shiga-like toxin II operon in Escherichia coli.Infection and Immunity 64:495.

Nakayama, K., S. M. Kelley, and R. Curtiss III. 1988. Construction of anAsd.sup.+expression-cloning vector: stable maintenance and high levelexpression of cloned genes in a Salmonella vaccine strain.Bio/Technology 6: 693–697.

Nakayama, K., S. M. Kelley, and R. Curtiss III. 1988. Construction of anAsd.sup.+expression-cloning vector: stable maintenance and high levelexpression of cloned genes in a Salmonella vaccine strain.Bio/Technology 6:693.

Nelson, S., S. E. Richardson, C. A. Lingwood, M. Petric, and M. A.Karmali. 1994. Biological activity of verocytotoxin (VT)2c and VT1/VT2cchimeras in the rabbit model. In Recent advances inverocytotoxin-producing Escherichia Coli infections. M. A. Karmali andA. G. Goglio, eds. Elsevier Science, New York, p. 245.

Niki, H., and S. Hiraga. 1997. Subcellular distribution of activelypartitioning F plasmid during the cell division cycle of E. coli. Cell90: 951–957.

Nordstrom, K. and S. J. Austin. 1989. Mechanisms that contribute to thestable segregation of plasmids. Annual Reviews in Genetics 23:37.

Noriega, F. R., G. Losonsky, J. Y. Wang, S. B. Formal, and M. M. Levine.1996. Further characterization of .DELTA.aroA .DELTA.virG Shigellaflexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as alive-vector vaccine for delivering antigens of enterotoxigenicEscherichia coli. Infect Irmun 64: 23–27.

Norioka, S., G. Ramakrishnan, K. Ikenaka, and M. Inouye. 1986.Interaction of a transcriptional activator, OmpR, with reciprocallyosmoregulated genes, ompF and ompC, of Escherichia coli. Journal ofBiological Chemistry 261:17113–17119

Nyholm, P., G. Magnusson, Z. Zheng, R. Norel, B. Binnington-Boyd, and C.A. Lingwood. 1996. Two distinct binding sites for globotriaosyl ceramideon verotoxins: identification by molecular modelling and confirmationusing deoxy analogues and a new glycolipid receptor for all verotoxins.Chemistry and Biology 3:263.

Nyholm, P., J. L. Brunton, and C. A. Lingwood. 1995. Modelling of theinteraction of verotoxin-1 (VT1) with its glycolipid receptor,globotriaosylceramide (Gb.sub.3). International Journal of BiologicalMacromolecules 17:199.

O'Brien, A. D. 1982. Innate resistance of mice to Salmonella typhiinfection. Infection and Immunity 38:948.

O'Brien, A. D., V. L. Tesh, A. Donohue-Rolfe, M. P. Jackson, S. Olsnes,K. Sandvig, A. A. Lindberg, and G. T. Keusch. 1992. Shiga toxin:biochemistry, genetics, mode of action, and role in pathogenesis.Current Topics in Microbiology and Immunology 180:65.

Olitsky, P. K. and I. J. Kligler. 1920. Toxins and antitoxins ofBacillus dysenteriae Shiga. Journal of Experimental Medicine 31:19.

Orosz, A., I. Boros, and P. Venetianer. 1991. Analysis of the complextranscription termination region of the Escherichia coli rrnB gene.European Journal of Biochemistry 201:653.

Oxer, M. D., C. M. Bentley, J. G. Doyle, T. C. Peakman, I. G. Charles,and A. J. Makoff. 1991. High level heterologous expression in E. coliusing the anaerobically-activated nirB promoter. Nucleic Acids Research19:2889–2892.

Pallen, M. J. and B. W. Wren. 1997. The HtrA family of serine proteases.Molecular Microbiology 26:209.

Pecota, D. C., C. S. Kim, K. Wu, K. Gerdes, and T. K. Wood. 1997.Combining the hoklsok, parDE, and pnd postsegregational killer loci toenhance plasmid stability. Applied and Environmental Microbiology63:1917–1924.

Perera, L. P., J. E. Samuel, R. K. Holmes, and A. D. O'Brien. 1991.Mapping the minimal contiguous gene segment that encodes functionallyactive Shiga-like toxin II. Infection and Immunity 59:829.

Perera, L. P., J. E. Samuel, R. K. Holmes, and A. D. O'Brien. 1991.Identification of three amino acid residues in the B subunit of Shigatoxin and Shiga-like toxin type II that are essential for holotoxinactivity. Journal of Bacteriology 173:1151.

Pittard, A. J. 1996. Biosynthesis of the aromatic amino acids. InEscherichia coli and Salmonella: Cellular and molecular biology. 2nd ed.F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B.Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.Umbarger, eds. ASM Press, Washington, D.C. p. 458.

Polisky, B. 1986. Replication control of the CoIE1-type plasmids. InMaximizing gene expression. W. S. Reznikoff and L. Gold, eds.Butterworths, Boston, p. 143.

Porter, R. D., S. Black, S. Pannuri, and A. Carlson. 1990. Use of theEscherichia coli ssb gene to prevent bioreactor takeover by plasmidlesscells. Bio/Technology 8:47.

Pouwels, P. H., R. J. Leer, M. Shaw, M. J. Heijne den Bak-Glashouwer, F.D. Tielen, E., Smit, B. Martinez, J. Jore, and P. L. Conway. 1998.Lactic acid bacteria as antigen delivery vehicles for oral immunizationpurposes. Int J Food Microbiol 41: 155–167.

Pratt, L. A., W. Hsing, K. E. Gibson, and T. J. Silhavy. 1996. Fromacids to osmZ: mutiple factors influence synthesis of the OmpF and ompCporins in Escherichia coli. Molecular Microbiology 20:911.

Puente, J. L., V. Alvarez-Scherer, G. Gosset, and E. Calva. 1989.Comparative analysis of the Salmonella typhi and Escherichia coli ompCgenes. Gene 83:197.

Richardson, S. E., T. A. Rotman, V. Jay, C. R. Smith, L. E. Becker, M.Petric, N. F. Olivieri, and M. A. Karmali. 1992. Experimentalverocytotoxemia in rabbits. Infection and Immunity 60:4154.

Ringquist, S., S. Shinedling, D. Barrick, L. Green, J. Binkley, G. D.Stormo, and L. Gold. 1992. Translation initiation in Escherichia coli:sequences within the ribosome-binding site. Molecular Microbiology6:1219.

Roberts, M., S. Chatfield, and G. Dougan. 1994. Salmonella as carriersof heterologous antigens. In Novel delivery systems for oral vaccines.D. T. O'Hagan, ed. CRC Press, Ann Arbor, p. 27–58.

Ruiz-Echevarria, M. J., G. Gimenez-Gallego, R. Sabariegos-Jareno, and R.Diaz-Orejas. 1995. Kid, a small protein of the parD stability system ofplasmid R1, is an inhibitor of DNA replication acting at the initiationof DNA synthesis. J Mol Biol 247: 568–577.

Rupp, W. D. 1996. DNA repair mechanisms. In Escherichia coli andSalmonella: Cellular and molecular biology. 2nd ed. F. C. Neidhardt, R.Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter and H. E. Umbarger, eds. ASMPress, Washington,D.C. p. 2277.

Ryan, E. T., J. R. Butterton, R. N. Smith, P. A. Carroll, T. I. Crean,and S. B. Calderwood. 1997a. Protective immunity against Clostridiumdifficile toxin A induced by oral immunization with a live, attenuatedVibrio cholerae vector strain. Infect Immun 65: 2941–2949.

Ryan, E. T., J. R. Butterton, T. Zhang, M. A. Baker, S. L. J. Stanley,and S. B. Calderwood. 1997b. Oral immunization with attenuated vaccinestrains of Vibrio cholerae expressing a dodecapeptide repeat of theserine-rich Entamoeba histolytica protein fused to the cholera toxin Bsubunit induces systemic and mucosal antiamebic and anti-V. choleraeantibody responses in mice. Infect Immun 65: 3118–3125.

Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: ALaboratory Manual, 2nd edition. Cold Spring Harbor, New York: ColdSpring Harbor Laboratory Press.

Selzer, G., T. Som, T. Itoh, and J. Tomizawa. 1983. The origin ofreplication of plasmid p15A and comparative studies on the nucleotidesequences around the origin of related plasmids. Cell 32:119.

Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993. Moleculargenetics of amino-glycoside resistance genes and familial relationshipsof the aminoglycoside-modifying enzymes. Microbiol Rev 57: 138–163.

Siegler, R. L. 1995. The hemolytic uremic syndrome. Pediatric Nephrology42:1505.

Siegler, R. L., A. T. Pavia, R. D. Christofferson, and M. K. Milligan.1994. A 20-year population-based study of postdiarrheal hemolytic uremicsyndrome in Utah. Pediatrics 94:35.

Sixma, T. K., P. E. Stein, W. G. Hol, and R. J. Read. 1993. Comparisonof the B-pentamers of heat-labile enterotoxin and verotoxin-1: twostructures with remarkable similarity and dissimilarity. Biochemistry32:191.

Srinivasan, J., S. A. Tinge, R. Wright, J. C. Herr, and R. Curtiss III.1995. Oral immunization with attenuated Salmonella expressing humansperm antigen induces antibodies in serum and the reproductive tract.Biology of Reproduction 53:462.

Stein, P. E., A. Boodhoo, G. J. Tyrrell, J. L. Brunton, and R. J. Read.1992. Crystal structure of the cell-binding B oligomer of verotoxin-1from E. coli. Nature 355:748.

Stoker, N. G., N. F. Fairweather, and B. G. Spratt. 1982. Versatilelow-copy-number plasmid vectors for cloning in Escherichia coli. Gene18: 335–341.

Streaffield, S. J., M. Sandkvist, T. K. Sixma, M. Bagdasarian, W. G.Hol, and T. R. Hirst. 1992. Intermolecular interactions between the Aand B subunits of heat-labile enterotoxin from Escherichia coli promoteholotoxin assembly and stability in vivo. Proceedings of the NationalAcademy of Sciences USA 89:12140.

Strockbine, N. A., L. R. M. Marques, J. W. Newland, H. W. Smith, R. K.Holmes, and A. D. O'Brien. 1986. Two toxin-converting phages fromEscherichia coli O157:H7 strain 933 encode antigenically distinct toxinswith similar biologic activities. Infection and Immunity 53:135.

Strockbine, N. A., M. P. Jackson, L. M. Sung, R. K. Holmes, and A. D.O'Brien. 1988. Cloning and sequencing of the genes for Shiga toxin fromShigella dysenteriae Type 1. Journal of Bacteriology 170:1116.

Strugnell, R. A., D. Maskell, N. F. Fairweather, D. Pickard, A.Cockayne, C. Penn, and G. Dougan. 1990. Stable expression of foreignantigens from the chromosome of Salmonella typhimurium vaccine strains.Gene 88: 57–63.

Summers, D. K. The Biology of Plasmids, 65–91, 1996.

Summers, D. K. 1998. Timing, self-control and sense of direction are thesecrets of multicopy plasmid stability. Mol Microbiol 29: 1137–1145.

Summers, D. K. and D. J. Sherratt. 1984. Multimerization of high copynumber plasmids causes instability: CoIE1 encodes a determinantessential for plasmid monomerization and stability. Cell 36:1097.

Tacket, C. O., D. M. Hone, R. Curtiss III, S. M. Kelly, G. L s nsky, L.Gu rs, A. M. Harris, R. Edelman, and M. M. Levine. 1992. Comparison ofthe safety and immunogenicity of .DELTA.aroC.DELTA.aroD and.DELTA.cya.DELTA.crp Salmonella typhi strains in adult volunteers.Infection and Immunity 60:536.

Tacket, C. O., M. Sztein, G. Losonsky, S. S. Wasserman, J. P. Nataro, R.Edelman, D. Pickard, G. Dougan, S. Chatfield, and M. M. Levine. 1997.Safety of live oral Salmonella typhi vaccine strains with deletions inhtrA and aroC aroD and immune responses in humans. Infection andImmunity 65:452–456.

Tacket, C. O., S. M. Kelley, F. Schodel, G. Losonsky, J. P. Nataro, R.Edelman, M. M. Levine, and R. Curtiss III. 1997. Safety andimmunogenicity in humans of an attenuated Salmonella typhi vaccinevector strain expressing plasmid-encoded hepatitis B antigens stabilizedby the Asd-balanced lethal vector system. Infection and Immunity65:3381–3385.

Takeda, Y. 1995. Shiga and Siga-like (Vero) toxins. In Bacterial toxinsand virulence factors in disease. J. Moss, B. Iglewski, M. Vaughan andA. Tu, eds. Marcel Dekker, Inc. New York, p. 313.

Tauxe, R. V. 1998. Public health perspective on immunoprophylacticstrategies for Escherichia coli O157:H7: who or what would we immunize?In Escherichia coli O157:H7 and other Shiga toxin-producing E. colistrains. J. B. Kaper and A. D. O'Brien, eds. ASM Press, Washington, D.C.In press.

Tesh, V. L., J. A. Burris, J. W. Owens, V. M. Gordon, E. A. Wadolkowski,A. D. O'Brien, and J. E. Samuel. 1993. Comparison of the relativetoxicities of Shiga-like toxins type I and type II for mice. Infectionand Immunity 61:3392.

Thisted, T., A. K. Nielsen, and K. Gerdes. 1994. Mechanism ofpost-segregational killing: translation of Hok, SmB and Pnd mRNAs ofplasmids R1, F and R483 is activated by 3′-end processing. EMBO Journal13:1950.

Thisted, T., N. S. Sorensen, and K. Gerdes. 1995. Mechanism ofpost-segregational killing: secondary structure analysis of the entireHok mRNA from plasmid R1 suggests a fold-back structure that preventstranslation and antisense RNA binding. Journal of Molecular Biology247:859.

Thisted, T., N. S. Sorensen, E. G. Wagner, and K. Gerdes. 1994.Mechanism of post-segregational killing: Sok antisense RNA interactswith Hok mRNA via its 5′-end single-stranded leader and competes withthe 3′-end of Hok mRNA for binding to the mok translational initiationregion. EMBO Journal 13:1960.

Tinge, S. A. and R. Curtiss III. 1990. Conservation of Salmonellatyphimurium virulence plasmid maintenance regions among Salmonellaserovars as a basis for plasmid curing. Infection and Immunity 58:3084.

Tinge, S. A. and R. Curtiss III. 1990. Isolation of the replication andpartitioning regions of the Salmonella typhimurium virulence plasmid andstabilization of heterologous replicons. Journal of Bacteriology 35172:5266.

Twigg, A. J., and D. Sherratt. 1980. Trans-complementable copy-numbermutants of plasmid CoIE1. Nature 283: 216–218.

Umbarger, H. E. 1978. Amino acid biosynthesis and its regulation. AnnualReviews in Biochemistry 47:533.

Valdivia, R. H. and S. Falkow. 1997. Fluorescence-based isolation ofbacterial genes expressed within host cells. Science 277:2007.

Valdivia, R. H., A. E. Hromockyj, D. Monack, L. Ramakrishnan, and S.Falkow. 1996. Applications for green fluorescent protein (GFP) in thestudy of host-pathogen interactions. Gene 173:47.

Van Melderen, L., P. Bernard, and M. Couturier. 1994. Lon-dependentproteolysis of CcdA is the key control for activation of CcdB inplasmid-free segregant bacteria. Mol Microbiol 11: 1151–1157.

Vicari, G., A. J. Olitzki, and Z. Olitzki. 1960. The action of thethermolabile toxin of Shigella dysenteriae on cells cultivated in vitro.British Journal of Experimental Pathology 41:179.

Wada, K., Y. Wada, F. Ishibashi, T. Gojobori, and T. Ikemura. 1992.Codon usage tabulated from the GenBank genetic sequence data. NucleicAcids Research 20:2111.

Wadolkowski, E. A., L. M. Sung, J. A. Burris, J. E. Samuel, and A. D.O'Brien. 1990. Acute renal tubular necrosis and death of mice orallyinfected with Escherichia coli strains that produce Shiga-like toxintype II. Infection and Immunity 58:3959.

Wahle, E., and A. Kornberg. 1988. The partition locus of plasmid pSC101is a specific binding site for DNA gyrase. EMBO J 7: 1889–1895.

Wang, S. and T. Hazelrigg. 1994. Implications for bcd mRNA localizationfrom spatial distribution of exu protein in Drosophila oogenesis. Nature369:400.

Wang, Y., Z. Zhang, S. Yang, and R. Wu. 1992. Cloning of par region andthe effect ofpar region on the stability of pUC9. Chinese Journal ofBiotechnology 8:107.

Williams, K. R., J. B. Murphy, and J. W. Chase. 1984. Characterizationof the structural and functional defect in the Escherichia colisingle-stranded DNA binding protein encoded by the ssb-1 mutant gene.Journal of Biological Chemistry 259:11804.

Wu, K., and T. K. Wood. 1994. Evaluation of the hok/sok killer locus forenhanced plasmid stability. Biotechnol Bioeng 44: 912–921.

Yamasaki, S., M. Furutani, K. Ito, K. lgarashi, M. Nishibuchi, and Y.Takeda. 1991. Importance of arginine at postion 170 of the A subunit ofVero toxin 1 produced by enterohemorrhagic Escherichia coli for toxinactivity. Microbial Pathogenesis 11:1.

Yanofsky, C., T. Platt, I. P. Crawford, B. P. Nichols, G. E. Christie,H. Horowitz, M. Van Cleemput, and A. M. Wu. 1981. The completenucleotide sequence of the tryptophan operon of Escherichia coli.Nucleic Acids Res 9: 6647–6668.

Yu, J. and J. B. Kaper. 1992. Cloning and characterization of the eaegene of enterohaemorrhagic Escherichia coli. Molecular Microbiology6:411.

Zalkin, H. and P. Nygaard. 1996. Biosynthesis of purine nucleotides. InEscherichia coli and Salmonella: Cellular and molecularbiology. 2nd ed.F. C. Neidhardt, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik,W. S. Reznikoff, M. Riley, M. Schaechter and H. E. Umbarger, eds. ASMPress, Washington, D.C. p. 561.

Zhang, X., Y. Lou, M. Koopman, T. Doggett, K. S. K. Tung, and R. CurtissIII. 1997. Antibody responses and infertility in mice following oralimmunization with attenuated Salmonella typhimurium expressingrecombinant murine ZP3. Biology of Reproduction 56:33.

Zoja, C., D. Corna, C. Farina, G. Sacchi, C. A. Lingwood, M. P. Doyle,V. V. Padhye, M. Abbate, and G. Remuzzi. 1992. Verotoxin glycolipidreceptors determine the localization of microangiopathic process inrabbits given verotoxin-1. Journal of Laboratory and Clinical Medicine120:229.

Zurita, M., F. Bolivar, and X. Soberon. 1984. Construction andcharacterization of new cloning vehicles VII. Construction of plasmidpBR327par, a completely sequenced, stable derivative of pBR327containing the par locus of pSC101. Gene 28:119.

1. A method for eliciting an immune response in a subject comprisingadministering a live attenuated bacterial vector vaccine to saidsubject, wherein the live attenuated bacterial vector vaccine comprisesan isolated cell comprising an expression vector, wherein saidexpression vector comprises a nucleotide sequence encoding: arestricted-copy-number origin of replication cassette comprising (i) anucleotide sequence encoding an origin of replication that limits theexpression vector to an average plasmid copy number of about 2 to 75copies per cell, (ii) a first unique restriction enzyme cleavage sitelocated 5′ of the nucleotide sequence encoding the origin ofreplication, and (iii) a second unique restriction enzyme cleavage sitelocated 3′ of the nucleotide sequence encoding the origin ofreplication; at least one post-segregational killing cassette comprising(i) a nucleotide sequence encoding at least one post-segregationalkilling locus, (ii) a first unique restriction enzyme cleavage sitelocated 5′ of the nucleotide sequence encoding the at least onepost-segregational killing locus, and (iii) a second unique restrictionenzyme cleavage site located 3′ of the nucleotide sequence encoding theat least one post-segregational killing locus, wherein said nucleotidesequence encoding at least one post-segregational killing locus encodesthe ssb post-segregational killing locus; and at least one partitioningcassette comprising (i) a nucleotide sequence encoding at least onepartitioning function, (ii) a first unique restriction enzyme cleavagesite 5′ of the nucleotide sequence encoding the at least onepartitioning function, and (iii) a second unique restriction enzymecleavage site located 3′ of the nucleotide sequence encoding the atleast one partitioning function.
 2. The method of claim 1, wherein therestricted-copy-number origin of replication is selected from the groupconsisting of: oriE1 (nucleotides 1250 to 1936 of SEQ ID NO: 1), ori101(nucleotides 50 to 2004 of SEQ ID NO: 3), and ori15A (nucleotides 50 to684 of SEQ ID NO: 2).
 3. The method of claim 1, wherein the averageplasmid copy-number falls within the range of about 5 to about 60 copiesper cell.
 4. The method of claim 1, wherein the partitioning function isan active partitioning function.
 5. The method of claim 1, wherein thenucleotide sequence encoding at least one partitioning functioncomprises parA.
 6. The method of claim 1, wherein the partitioningfunction is a passive partitioning function.
 7. The method of claim 1,wherein the nucleotide sequence encoding at least one partitioningfunction is the par locus of pSC101.
 8. The method of claim 1, furthercomprising an expression cassette comprising (i) a nucleotide sequenceencoding a promoter, (ii) a first unique restriction enzyme cleavagesite located 5′ of the nucleotide sequence encoding the promoter, and(iii) a second unique restriction enzyme cleavage site located 3′ of thenucleotide sequence encoding the promoter.
 9. The method of claim 1,wherein the bacterial strain is Salmonella typhi or a Salmonella typhistrain.
 10. The method of claim 1, wherein the subject is selected fromthe group consisting of a mammal, a human and a bovine.
 11. The methodof claim 8, wherein the promoter is an inducible promoter.
 12. Themethod of claim 8, further comprising a nucleotide sequence encoding anantigen of interest located at the 3′ end of the nucleotide sequenceencoding the promoter.
 13. The method of claim 11, wherein the promoteris an ompC promoter.
 14. The method of claim 13, wherein the ompCpromoter comprises a polynucleotide fragment spanning nucleotides +70through −389, relative to the transcriptional start site +1, of E. coliompC.
 15. The method of claim 13, wherein the ompC promoter comprisesthe following sequence: AGATCX₁X₂TAAX₃CATCCACAGGAGGATATCTGATG (SEQ IDNO: 36), wherein X₁ is selected from the group consisting of G, C and A;X₂ is an insert having from 1 to 5 nucleotides; and X₃ is selected fromthe group consisting of A, T, G and C.
 16. The method of claim 15,wherein X₁ is G.
 17. The method of claim 15, wherein X₂ has from 1 to 4nucleotides.
 18. The method of claim 15, wherein X₂ has 4 nucleotides.19. The method of claim 15, wherein X₂ has 4 nucleotides, independentlyselected from the group consisting of A, T and C.
 20. The method ofclaim 15, wherein X₂ comprises a nucleotide or nucleotide sequenceselected from the group consisting of ATCT; ATC; AT; TCT; CT; TC; A; T;C; and T.
 21. The method of claim 15, wherein X₂ is selected from thegroup consisting of ATCT; ATC; AT; TCT; CT; TC; A; T; C; and T.
 22. Themethod of claim 15, wherein X₂ is ATCT.
 23. The method of claim 15,wherein X₃ is A.
 24. The method of claim 12, wherein the antigen ofinterest is selected from the group consisting of a viral antigen, abacterial antigen, a cancer antigen, and an auto-immune antigen.
 25. Themethod of claim 24, wherein the antigen of interest comprises adetoxified Shiga toxin.
 26. The method of claim 25, wherein the antigenof interest comprises a Shiga toxin 2 antigen selected from the groupconsisting of a Shiga toxin 2 B subunit pentamer and a geneticallydetoxified Shiga toxin
 2. 27. The method of claim 26, wherein the geneencoding the detoxified Shiga toxin 2 has modified segments selectedfrom the group consisting of: (SEQ ID NO: 37)  (797) - ACA GCA GAC GCGTTA - (811); (SEQ ID NO: 38)  (902) - CTG AAC CTA GGG CGA - (916); (SEQID NO: 39) (1345) - GAA TTC GCG ACC AGT - (1359) and (SEQ ID NO: 40)(1435) - GAA TCA GAT TCT GGA - (1449).


28. The method of claim 9, wherein the endogenous ssb gene of Salmonellatyphi or a Salmonella typhi strain is deleted.